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Yeast Protocols Handbook

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1. Figure 1 Sequence of the GAL4 DNA BD recognition sites in the GAL1 GAL2 and MEL1 UASs and the UASg 17 mer consensus sequence Giniger 8 Ptashne 1988 Clontech Laboratories Inc www clontech com Protocol No PT3024 1 6 Version No PR742227 Yeast Protocols Handbook ll Introduction to Yeast Promoters continued Heslot amp Gaillardin 1992 provides the binding site for the GAL4 DNA BD For LexA based systems multiple copies of the LexA operator provide the binding site for the LexA protein If you are putting together your own one or two hybrid system you must make sure that the reporter gene s promoter will be recognized by the DNA BD moiety encoded in your DNA BD fusion vector Reporter genes under the control of GAL4 responsive elements In yeast the genes required for galactose metabolism are controlled by two regulatory proteins GAL4 and GAL80 as well as by the carbon source in the medium Guthrie amp Fink 1991 Heslot amp Gaillardin 1992 When galactose is present the GAL4 protein binds to GAL4 responsive elements within the UAS upstream of several genes involved in galactose metabolism and activates transcription Inthe absence of galactose GAL80 binds to GAL4 and blocks transcriptional activation Furthermore in the presence of glucose transcription of the galactose genes is immediately repressed Johnston et al 1994 The UASs of the 20 known galactose responsive genes all con
2. e Thaw and resuspend cell pellets in cold TCA buffer e Add cells to glass beads and ice cold 20 TCA e Bead beat cells 2 x 30 sec or vortex vigorously for 10 min at 4 C Beads First Cell Extract and unbroken cells liquid above beads e Add ice cold 20 TCA e Bead beat cells 1 x 30 sec or vortex for 5 min at 4 C Beads Second Cell Extract and unbroken cells liquid above beads discard Combined Cell Extracts liquid above beads e Place on ice e Combine Cell Extracts e Allow glass beads to settle 1 min Beads and unbroken cells discard e Centrifuge at 14 000 rpm for 10 min Pellet Protein and contaminants Supernatant Discard e Resuspend in TCA Laemmli loading buffer e Boil 10 min e Centrifuge at 14 000 rpm for 10 min Supernatant gt Immediately load gel or Pellet discard Protein extract freeze at 70 C or colder Figure 3 TCA protein extraction method Clontech Laboratories Inc www clontech com Protocol No PT3024 1 16 Version No PR742227 Yeast Protocols Handbook IV Preparation of Yeast Protein Extracts continued E Troubleshooting Optimal electrophoretic separation of proteins depends largely on the quality of the equipment and reagents used in the gel system the manner in which the protein samples are prepared prior to electrophoresis the amount of protein loaded on the gel and the voltage conditions used during electrophoresis Th
3. Chien C T Sternglanz R amp Fields S 1993a Using the two hybrid system to detect protein protein interactions In Cellular Interactions in Development A Practical Approach ed Hartley D A Oxford University Press Oxford pp 153 179 Bartel P L Chien C T Sternglanz R amp Fields S 1993b Elimination of false positives that arise in using the two hybrid system BioTechniques 14 920 924 Brent R 8 Ptashne M 1985 A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor Cell 43 729 736 Chien C T Bartel P L Sternglanz R 8 Fields S 1991 The two hybrid system A method to identify and clone genes for proteins that interact vvith a protein of interest Proc Nat Acad Sci USA 88 9578 9582 Fields S amp Song O 1989 A novel genetic system to detect protein protein interactions Nature 340 245 247 Fields S 1993 The two hybrid system to detect protein protein interactions METHODS A Companion to Meth Enzymol 5 116 124 Fields S amp Sternglanz R 1994 The two hybrid system an assay for protein protein interactions Trends Genet 10 286 292 Fritz C C amp Green M R 1992 Fishing for partners Current Biol 2 403 405 Golemis E A Gyuris J amp Brent R 1996 Analysis of protein interactions and Interaction trap two hybrid systems to identify interacting proteins In Current Protocols in Molecular Biology John Wiley amp
4. Incubate at 30 C with shaking 220 250 rpm until the OD reaches 0 4 0 6 Depending on the fusion protein this will take 4 8 hr Multiply the OD of a 1 ml sample by the culture volume i e 55 ml to obtain the total number of units this number will be used in Sections C amp D For example 0 6 x 55 ml 33 total OD oo units Note During late log phase the ADH7 promoter shuts down and the level of endogenous yeast proteases increases N 4 Quickly chill the culture by pouring it into a prechilled 100 ml centrifuge tube halfway filled with ice Immediately place tube in a prechilled rotor and centrifuge at 1000 x g for 5 min at 4 C 6 Pour off supernatant and resuspend the cell pellet in 50 ml of ice cold H O Any unmelted ice pours off with the supernatant Recover the pellet by centrifugation at 1 000 x g for 5 min at 4 C Immediately freeze the cell pellet by placing the tube on dry ice or in liquid nitrogen Store cells at 70 C until you are ready to proceed with the experiment o N Clontech Laboratories Inc www clontech com Protocol No PT3024 1 12 Version No PR742227 Yeast Protocols Handbook IV Preparation of Yeast Protein Extracts continued C Preparation of Protein Extracts Urea SDS Method Figure 2 Printen amp Sprague 1994 Reagents and Materials Required e 1 5 ml screw cap microcentrifuge tubes Glass beads 425 600 um Sigma Cat No G 8772 Protease
5. Minimal SD Base and Minimal SD Agar Base either with dextrose glucose or galactose raffinose are available from Clontech in powder form See Chapter XI for ordering information If you purchase Clontech s Minimal SD Base prepare your SD Dropout DO medium according to the instructions provided For example to prepare SD Leu Trp agar you will need to combine SD Minimal Agar Base Cat No 630412 with Leu Trp DO Supplement Cat No 630412 Alternatively you can purchase yeast nitrogen base from another supplier e g Difco Cat No 0919 15 3 and prepare SD DO medium as follows 6 7 g Yeast nitrogen base without amino acids 20 g Agar for plates only 850 ml H O 100 ml of the appropriate sterile 10X Dropout Solution e Adjust the pH to 5 8 if necessary and autoclave Allow medium to cool to 55 C before adding 3 AT cycloheximide additional adenine or X gal see below e Add the appropriate sterile carbon source usually dextrose glucose to 2 unless specified otherwise for your application Adjust the final volume to 1 L if necessary Notes Ifyou add the sugar solution before autoclaving autoclave at 121 C for 15 min autoclaving ata higher temperature foralonger period of time or repeatedly may cause the sugar solution to darken and will decrease the performance of the medium Note that SD Minimal Base from Clontech already contains a carbon source e If you purchase galactose separately it must be highly
6. 20 mM 20 ml of 1 M glucose Deionized H O to1 L Before adding MgCl MgSO and glucose stock solutions separately filter sterilize them using a 0 2 um filter Add the bactotryptone yeast extract and NaCl to 900 ml of deionized H O stir or shake until solutes have dissolved Add the KCI Adjust the pH to 7 with 5 N NaOH 0 2 ml Adjust the volume to 960 ml with deionized H O and autoclave Just before use add filter sterilized MgCl MgSO and glucose LB broth Bacto tryptone 10 g L Bacto yeast extract 5 g L NaCl 5 g L Adjust pH to 70 with 5 N NaOH Autoclave Store broth at 22 C LB amp agar plates Prepare LB broth Sambrook et al 1989 as above Add agar 18 g L autoclave and cool to 50 C Add ampicillin to 50 ug ml Pour plates and store at 4 C M9 minimal medium for nutritional selection of E coli transformants complemented by the wild type yeast gene For optimal recovery of KC8 and HB101 transformants add a 1X mixture of amino acids i e dropout DO supplement lacking the specific nutrient that will allow selection of the desired plasmid The same DO supplements used for yeast SD medium can be used to supplement M9 minimal medium see Appendix C A for dropout recipe or purchase premixed DO Supplements from Clontech In addition KC8 requires thiamine and HB101 requires thiamine and proline for growth on minimal medium Prepare 900 ml of M9 medium as directed in Sambrook et al 1989 To prepare agar pl
7. C for 10 min Note This initial incubation at 70 C frees membrane associated proteins Thus if you skip this step membrane associated proteins will be removed from the sample at Step 6 high speed centrifugation Vortex vigorously for 1 min Pellet debris and unbroken cells in a microcentrifuge at 14 000 rpm for 5 min preferably at 4 C otherwise at room temperature 20 22 C 7 Transfer the supernatants to fresh 1 5 ml screw cap tubes and place on ice first 8 supernatants Treat the pellets as follows a Place tubes in a 100 C boiling water bath for 3 5 min b Vortex vigorously for 1 min c Pellet debris and unbroken cells in a microcentrifuge at 14 000 rpm for 5 min preferably at 4 C otherwise at room temperature d Combine each supernatant second supernatant with the corresponding first supernatant from Step 7 Note If no supernatant is obtained add more cracking buffer 50 100 ul and repeat Steps 8 b amp c Boil the samples briefly Immediately load them on a gel Alternatively samples may be stored on dry ice or in a 70 C freezer until you are ready to run them on a gel Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 13 Yeast Protocols Handbook IV Preparation of Yeast Protein Extracts continued Cell pellets e Thaw and resuspend cell pellets in prewarmed Cracking buffer e Add cells to glass beads e Heat at 70 C for 10 min e Vo
8. Keep buffer on ice until you are ready to use it 3 Vortex the overnight culture tube for 0 5 1 minto disperse cell clumps Immediately transfer at least 2 ml of the overnight culture to no more than 8 ml of YPD except for the LexA System Note For the LexA System use the appropriate SD Gal Raff induction medium for the strains being assayed 4 Incubate the fresh culture at 30 C for 3 5 hr with shaking 230 250 rpm until the cells are in mid log phase OD of 1 ml 0 4 0 6 5 Vigorously vortex the culture tube for 0 5 1 min to disperse cell clumps Record the exact OD when you harvest the cells 6 Place 1 5 ml of culture into each of three 1 5 ml microcentrifuge tubes Centrifuge at 14 000 rpm 10 000 x g for 30 sec 7 Carefully remove supernatants Add 1 5 ml of Z buffer to each tube and thoroughly resuspend the pellet 8 Centrifuge at 14 000 rpm 10 000 x g for 30 sec 9 Remove the supernatants Resuspend each pellet in 300 ul of Z buffer Thus the concentration factor is 1 5 0 3 5 fold 10 Read the of the resuspended cells The OD should be 2 5 If the cell density is lower repeat Steps 5 9 except resuspend the cells in lt 300 ul of Z buffer 11 Vortex each cell suspension and transfer 100 ul to a fresh tube Note The remaining cell suspension can be stored at 70 C to 80 C 12 Place tubes in liquid nitrogen for 0 5 1 min to freeze the cells 13 Place frozen tubes in a 37 C water bath for
9. LTV TV 2941 VOS P vpleb 9661 75 18 s uuer Zn ein dij LTA 3GV ESIH 002 6914 ZG Eun ZLL zZn l L06 L dN ELVIN s Heiqr 14 iWC 69f d DSI gape VOS E VilP5 109 14 Zil Zn 106 1d4 HL ININ 661 7278 Ldn esiy gape LOL zape 108 28 1 002 8814 Z3 4n PIVW 3 HZ Vx91 VAZCVINA G66L 75 2 yelo sq gein d gsiq onal zn3v yx r esiy gen OLY 2 VX91 STA 4 Ze Ty5 TV vun LTAW S H ES H LTVO ZSAT 0661 uolsuuor 01 4 Z o VOS E 211 zn l 106 1d4 12661 1 d1eH znal idi LTAW 298 ESIH 108 25 1 LOL Z PE OOZ ESIY Z3 4n ELYN H Y Y O6LA Zoe OA g OPP p79 EVEN ESIN LTW9O S TWD ZSAT uolmegoiunuuuuoo jeuosad XNOIID 9 2 869 08156 209 186 211 Zn 106 10411 7661 8 19 19110l 94 znal 2291 ESIH L08 ZSA 101 2 002 6914 ZG E81N ELYN 2 2 Y Y 9961 0 1311 220 0 Seven pselvesqy Ld vog e6 jaw VplP5 Zil n 106 1 411 53 E HZ 6661 8 znal Ldn LTAW 229 101 2 OOT ESIY TG EBIN OLY Z HZ L8LA Zoe NHAI piy EVEN 291 0 1 vD ZSAT uoljediuNnWIWOd guosi d XNOIID 9 924 08188 2 9 1 211
10. Store at 4 C for up to 3 months Buffer 2 20 ml Dissolve 27 1 mg of CPRG in 20 ml of Buffer 1 final concentration of CPRG is 2 23 mM Filter sterilize Store at 4 C in the dark for up to 3 months F For o Gal Quantitative Assays PNP o Gal Solution 100 mM p nitrophenyl a p Galactopyranoside Sigma Cat No N0877 in deionized H O For 10 ml dissolve 301 3 mg of PNP a Gal in 10 ml of deionized H O Filter sterilize Notes Prepare solution fresh before each use Keep the p nitrophenyl a p Galactopyranoside solid anhydrous Store in a dessicator at 20 C 10X Stop Solution 1 M Na CO in deionized H O Sigma Cat No S7795 1X NaOAc 0 5 M sodium acetate pH 4 5 Sigma Cat No S7545 Assay Buffer Prepare Assay Buffer fresh before each use by combining 2 volumes 1X NaOAc Buffer with 1 volume PNP a Gal Solution 2 1 v v ratio Mix well Clontech Laboratories Inc www clontech com Protocol No PT3024 1 60 Version No PR742227 Yeast Protocols Handbook APPENDIX E Plasmid Information NS IPS e ute1uoo JOU s op OLAYDA eS YUM uons Bip Aq pazieaul si pZVAVDI p ZSVd ul anbiun si Ho33 ay u5931uo o9 18 paljipolw sem piluse d au 5141 u p q i5s p plulseld y JO AHBALH P e SI 1 ZSVd 5 S H BAQ 493puuuoS1e N pue 1e nB 1 YIM paplAosd sjaxoeg UONEWIOJU 4032 A pue JOS uu9 s S VxX871
11. Z UB LdHL 14800 u uo uolo I YOOF HWeg 62 dul 0062991 ule ueuunH HZ ue1 LyigSd 07 67 VEA ldu uo luol5 I Ho53 IHtueg 68 114800 u eed auunu HZ VIVD 29 714890 072 0 8 dwe 2131 LLAV9d ul 40914012 10023 1 04 001 02 78 UaBrue OVAS 6 2 FTV 1 10 00 dwe 2131 87 8 Z Z LL AHEAH P OGdDAe u 6861 Buos spl 4 III pu H 691 neq PIV u Bu l IIn d43 pilAA 2 2 8 HZ PIVD 119d L66L 2 19 u lu 470 90 610 O L UZ 776 due LdYL Ayzygd u 16661 spl 4 17 III pu H 98 80 8 ya6iUe 261e OVAS HZ Vx91 l avzvad vE 8LHSq 6 1 1Z 9 dwe eyyn lyxa 9661 e 12 yelols3 III pu H 601 ean JO J04 U09 19pun 228 HZ VX91 Zov dogd 9u 19 1 ul uueu 44 s s 3 H nsouBeiq qx ds uondu s q ui s s e1039 A s u l z s UO uon l s SGIINSW1d TOHLNO ANY 1 4 IWWALSAS 8 H V NH LV N HA 318V1 Protocol No PT3024 1 www clontech com Clontech Laboratories Inc 62 Version No PR742227 Yeast Protocols Handbook ion continued Plasmid Informati APPENDIX E Bg Aq in ou ae anjgegd pue SIHEGd Woy Wey usinBunsip o lqissod 11 s yeul YoIYyM alls ELS lBuls e ABU 1Z79e7d pue IS Hd ul 1W INOYUM SIH GS UO JOxJeLU lqe l s e se sn sli sjiluJed spiuu
12. a two different mutations are combined in a diploid cell or b when a wild type allele on a plasmid is introduced into a cell bearing a defective chromosomal allele via yeast mating or transformation genome The entire complement of genetic material in a cell excluding autonomously replicating plasmids and mitochondrial DNA genotype Generally a list of mutant alleles and exogenous genetic elements Wild type alleles are sometimes listed as well for clarity in a specific experimental context haploid A cell having one chromosome set A diploid cell or organism can also be haploid for a given gene due to chromosomal deletions hybridization probe A defined nucleic acid segment which can be labeled and used to identify specific DNA clones bearing the complementary sequence via hybridization leaky mutant A mutant that represents a partial rather than a complete inactivation of the wild type function leaky phenotypes can result from a mutation in the coding region or inthe promoter region In yeast one and two hybrid systems some of the host strains are leaky for expression of certain auxotrophic markers for example H S3 expression in Y190 mating types A genetically haploid state of unicellular organisms that can reproduce sexually by cellular and nuclear fusion to produce a diploid organism In S cerevisiae there are two mating types a and a which differ only physiologically and not in physical form mutant An organism or
13. should lie between 0 5 1 0 Dilute the cell suspension if necessary remember to account for the dilution factor when making your final calculations Step 11 below 4 Place 1 0 ml of culture into a 1 5 ml microcentrifuge tube Centrifuge at 14 000 rpm 10 000 x g for 2 min or until the cells are completely pelleted Note It is important to ensure that cells and cellular debris are completely pelleted to minimize intereference from light scattering in the colorimetric assay below 5 Carefully transfer the supernatant to a clean tube and store at room temperature for use in Step 6 below Note To minimize the loss of enzyme activity we suggest proceeding with the colorimetric assay immediately once the cell free superantant has been isolated Colorimetric Assay Belowwe provide protocols for 1 mland200 ul assays Ifyou have access to a spectrophotometer equipped to read microtiter plates you may find it more convenient to use the 200 ul assay protocol which is intended for use with 96 well flat bottom microtiter plates Note The experimental conditions and volumes of reagents used throughout the a Galactosidase Quanitative Assay have been carefully tested and optimized for use in the 1 ml and 200 ul assay formats described below Please follow as directed Though we do not recommend changing the actual volumes of reagents used in the colorimetric assay if the signal from an experimental sample exceeds the linear range of the ass
14. 1 www clontech com Clontech Laboratories Inc Version No PR742227 29 Yeast Protocols Handbook VI a and p Galactosidase Assays continued Optional Purified B galactosidase for a standard curve Note For best results we recommend using the Luminescent B galactosidase Detection Kit II Cat No 631712 which includesareaction buffercontainingthe Galacton Star substrate andthe Sapphirell accelerator positive control bacterial B galactosidase and a complete User Manual Chemiluminescent detection of f galactosidase Itis importantto stay within the linear range of the assay High intensity light signals can saturate the photomultiplier tube in luminometers resulting in false low readings In addition low intensity signals that are near background levels may be outside the linear range of the assay Ifin doubt determine the linear range of the assay and if necessary adjust the amountof lysate used to bring the signal within the linear range See Campbell et al 1995 for a chemiluminescent B galactosidase assay used in a yeast two hybrid experiment 1 Prepare 5 ml overnight cultures in liquid SD medium as described in 11 Use the SD medium appropriate for your system and plasmids Note For qualitative data a whole colony resuspended in Z buffer may be used for the assay directly See instructions following this section 2 On the day of the experiment prepare the Galacton Star reaction mixture
15. A See Sambrook et al 1198911 1 22 1 23 e Boiling lysis is not recommended for isolation of plasmids from endA bacteria e If you are using a commercial plasmid preparation kit follow the manufacturer s directions for host strains that are endA e If you plan to use the plasmid for sequencing or other applications requiring highly purified DNA the plasmid should be extracted with phenol chloroform isoamyl alcohol and precipitated with ethanol before use Alternatively CHROMA SPIN TE 400 Columns Cat No K1323 1 may be used to purify the plasmid b To verify that you have obtained the correct plasmid amplify the insert by PCR digest it with Hae Ill and run a small sample on an agarose EtBr gel Compare the restriction digestion pattern with that of the original clone isolated from yeast 4 Troubleshooting tips a If you do not obtain any transformants you may need to improve the transformation effiency of the cells e f you performed a nutritional selection on M9 minimal medium repeat the transformation but plate the cells on LB amp instead The recovery of new transformants is generally better on on LB amp than on M9 medium Then replica plate the Amp transformants to the appropriate M9 minimal medium for selection of the desired plasmid and to verify that the undesired plasmid s have been lost Note that it takes somewhat longer to see colonies on M9 medium than on LB If you are not already doin
16. Galactosidase Assays continued TABLE III COMPARISON OF B GALACTOSIDASE ASSAYS Protocol Type of assay Substrate Section Applications Comments In vivo X gal in medium VI B e Less sensitive than colony lift assays agar plate recommended only vvhen the cellsto be assayed contain many copies of the lacZ reporter gene such as on a high copy number plasmid Convenient for large scale experiments screen many plates and colonies at the same time e Potential drawbacks Qualitative results only Expensive if assaying many plates Need to check for blue color development at several time intervals between 24 and 96 hr Background can be troublesome Colony lift X gal on filter VI C e Relatively sensitive recommended when the cells filter to be assayed contain one or only afew copies of the lacZ reporter gene Convenient for large scale experiments screen many plates and colonies at the same time e Relatively inexpensive to screen many plates e Get results quickly in most cases within a few hours e Potential drawbacks Qualitative results only More manipulations required than for in vivo assay Liquid culture ONPG VI D e Forassaying asmallnumber ofselectedtransformants e Less expensive than CPRG or Galacton Star e Potential drawbacks May not be sensitive enough to quantify weak or transient two hybrid interactions Liquid culture CPRG VILE e Forassaying asmallnumber ofselectedtransformants e 10 times more s
17. Materials Required e Appropriate SD agar plates that will keep selection on the plasmid s of interest Appendix E or the system specific User Manual Labeled cDNA probe complementary to previously isolated cDNAs Note Oligonucleotides random primed cDNAs or PCR generated fragments can be used as probes Oligonucleotide probes may be advantageous if the cDNA is a member of a protein family to avoid inadvertently excluding related genes that are not identical to those initially obtained 1 M sorbitol 20mM EDTA 50 mM DTT prepare fresh 1 M sorbitol 20 mM EDTA 0 5 M NaOH 0 5 M Tris HCI pH 75 6X SSC Ausubel et al 1994 2X SSC Ausubel et al 1994 100 000 units ml B glucuronidase type HP 2 crude solution from Helix pomatia Sigma Cat No G 7017 82 mm circular nylon membrane sterile Whatman 3 MM paper 80 C vacuum oven or UV cross linker Additional reagents and equipment for bacterial filter hybridization Ausubel et a 1994 If you have not done so already collect the colonies to be screened onto a master plate in a grid pattern to facilitate future identification of the colonies Include a positive and negative control on each plate Since this will be your master plate it is important to use the appropriate SD agar medium to maintain selection on all plasmids including any reporter plasmid Incubate plate at 30 C for 2 4 days until colonies appear 2 Prepare sorbitol EDTA DTT solution 3 For each plate
18. Note Before checking the OD vortex the culture tube for 0 5 1 min to disperse cell clumps 4 Place 1 5 ml of culture into each of three 1 5 ml microcentrifuge tubes Centrifuge at 14 000 rpm 16 000 x g for 30 sec to pellet the cells 5 Carefully remove the supernatant add 1 0 ml of Buffer 1 and vortex until cells are thoroughly resuspended 6 Centrifuge at 14 000 rpm 16 000 x g for 30 sec to pellet the cells Carefully remove the supernatant and resuspend the cells in 300 ul of Buffer 1 The concentration factor is 1 5 0 3 5 fold Note Differences in cell recoveries after this wash step can be corrected for by re reading the OD of the resuspended cells 8 Transfer 0 1 ml of the cell suspension to a fresh microcentrifuge tube 9 Place tubes in liquid nitrogen until the cells are frozen 0 5 1 min 10 Place frozen tubes in a 37 C water bath for 0 5 1 min to thaw 11 Repeat the freeze thaw cycle Steps 9 and 10 two times to ensure that all cells are broken open 12 Add 0 7 ml of Buffer 2 to each sample and mix by vortexing Thorough mixing is critical to the assay 13 Record the time when Buffer 2 was added This is the starting time 14 Add 1 ml of Buffer 2 to a separate tube this will be the buffer blank 15 When the color of the samples is yellow grey to red add 0 5 ml of 3 0 mM ZnCl to each sample andthe buffer blankto stop color development Record the stoptime For very strong B galactosi
19. PCR amplification using AD vector specificinsert screening primers Reagents and materials required E coli competent cells chemically competent or electrocompetent Notes For methods to prepare electrocompetent E coli cells see Kaiser amp Auer 1993 Dower et al 1988 Chuang et al 1995 and Sambrook etal 1989 Alternatively purchase premade chemically competent or electrocompetent E coli cells from Clontech If you use the direct electroporation method of Marcil amp Higgins 1992 the E coli competent cells must be transformed at an efficiency of 109 cfu ug of pUC19 DNA to work satisfactorily with yeast plasmids e For transformation of electrocompetent cells you need an electroporator and a cuvette with a 0 1 cm gap Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook VII Working With Yeast Plasmids continued e Yeast plasmid DNA from Section B above Sterile 14 ml polypropylene conical tubes e g Falcon Cat No 2059 e Hanahan s SOC medium or LB broth Sambrook et al 1989 LB amp 50 ug ml agar plates for antibiotic selection or appropriately supplemented M9 amp plates for nutritional selection Appendix C B e Materials for isolating plasmid DNA from E coli 1 Procedure for transforming electrocompetent E coli KC8 a Prepare or thaw electrocompetent E coli cells b Add 1 2 ul of yeast plasmid solution
20. Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 43 Yeast Protocols Handbook IX Additional Useful Protocols continued c Incubate the plate at 30 C for 2 3 days or until colonies appear d Using sterile toothpicks or pipette tips transfer 20 30 individual colonies in an orderly grid fashion to appropriate SD selection plates to verify that they have lost the unwanted plasmid and retained the plasmid of interest Note Store the yeast segregants on the appropriate SD selection plates wrapped in Parafilm at 4 C for up to two weeks 2 Cycloheximide counterselection of yeast segregants Some yeast host strains such as CG 1945 and Y190 carry the cyh 2 mutant allele and are cycloheximide resistant Cyh C Giroux personal communication for CG 1945 and Harper et al 1993 for Y190 The wild type CYH 2 gene is dominant to the cyh 2 mutant allele Thus when transformed with a plasmid such as pAS2 1 that contains the wild type CYHS2 gene the host strain will become sensitive to cycloheximide this holds true for a Cyh host strain cotransformed with a CYH 2 bearing plasmid and another plasmid that does not carry the CYH 2 gene Therefore one can effectively select for yeast cells that have spontaneously lost the CYH 2 bearing plasmid while retaining the other plasmid simply by plating the cotransformants on the appropiate SD medium containing cycloheximide Note The CYH2 gene encodes
21. Protocols Handbook a and 6 Galactosidase Assays continued 4 Using forceps place a clean dry filter over the surface of the plate of colonies to be assayed Gently rub the filter with the side of the forceps to help colonies cling to the filter 5 Poke holes through the filter into the agar in three or more asymmetric locations to orient the filter to the agar 6 When the filter has been evenly wetted carefully lift it off the agar plate with forceps and transfer it colonies facing up to a pool of liquid nitrogen Using the forceps completely submerge the filters for 10 sec Note Liquid nitrogen should be handled with care always wear thick gloves and goggles 7 After the filter has frozen completely 10 sec remove it from the liquid nitrogen and allow it to thaw at room temperature This freeze thaw treatment is to permeabilizes the cells 8 Carefully place the filter colony side up on the presoaked filter from Step C 3 Avoid trapping air bubbles under or between the filters 9 Incubate the filters at 30 C or room temperature and check periodically forthe appearance of blue colonies Notes The time it takes colonies producing 8 galactosidase to turn blue varies typically from 30 min to 8 hr ina library screening Prolonged incubation gt 8 hr may give false positives Yeast transformed with the B galactosidase positive control plasmid will turn blue within 20 30 min Most yeast report
22. Zn LOG L 41 7661 Je 19 1 VO0 4 znal dn Zel ESIH LO8 ZSA 101 2 OOZ ESIY ZG 4n ELYN HZ 1 Z4H Zoe 1 Typ V Ty even 864 08186 2 04 1 upo Z L gn 106 1 dn 2661 2 19dieH n 229 108 29 101 20 OOZ ESIY ZG 4n el VW HZ 928 49 s 5u 1 J t SH YHEH N 5 e dA ou n 5 ulens uoneuuojJsue4 SINALSAS GNV 4NO H3yIVIAHO1V A 3H1 NI SNIVYLS MalMOd3au 15 XI 318V1 Protocol No PT3024 1 www clontech com Clontech Laboratories Inc 64 Version No PR742227 Yeast Protocols Handbook Notes AmpliTaq and GeneAmp are registered trademarks of Hoffmann LaRoche Inc and F Hoffmann LaRoche Ltd Falcon is a trademark of Becton Dickinson amp Co Galacton Star and Sapphire II are trademarks of Tropix Inc GenBank is a trademark of the U S Department of Health amp Human Services Parafilm is a registered trademark of the American Can Co Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 65 Yeast Protocols Handbook Notes Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227
23. cell carrying a mutation Clontech Laboratories Inc www clontech com Protocol No PT3024 1 50 Version No PR742227 Yeast Protocols Handbook APPENDIX A Glossary of Technical Terms continued mutant allele An allele differing from the allele found in the standard or wild type mutation a The process that produces a gene or a chromosome differing from the wild type b The DNA or amino acid change resulting from such a process operator In bacteria a DNA region that acts a binding site for a specific repressor protein and thereby exerts control over transcription of the adjacent structural gene or operon operon In bacteria a set of adjacent structural genes that are transcribed into a single mRNA molecule plus the adjacent regulatory genes that affect transcription of the structural genes PCR Polymerase chain reaction a process by which a defined segment of DNA is exponentially replicated in vitro by the action of a thermostable DNA polymerase during repeated cycles of heating and cooling phenotype The observable properties of an organism determined by the organism s genetic constitution genotype and the effects of the environment plasmid A genetic element in bacteria or yeast that can replicate autonomously in the host cell Some plasmids can also be inserted into the host s genome in defined natural or experimental situations e g via transformation of linearized plasmid DNA promoter A DNA sequence to wh
24. characterizing PCR products a Electrophorese 10 ul samples of the PCR product on an EtBr 0 8 agarose gel to confirm that the PCR worked and to determine if the plasmid prep contains multiple nonhomogeneous plasmids b Digest another 10 ul sample of each amplified insert with a frequent cutter restriction enzyme such as Alu l or Hae Ill in a 20 ul volume reaction Run these samples on an EtBr 1 8 agarose gel in parallel with DNA size markers for comparison 7 Good PCR practices a Prepare reactions with dedicated pipettors in a dedicated work space Due tothetremendous amplification power of PCR minute amounts of contaminating DNA can produce nonspecific amplification in some instances contaminants can cause DNA bands even in the absence of added template DNA We recommend that you set up your PCR reactions in a dedicated lab area or noncirculating containment hood and use dedicated pipettors PCR pipette tips with hydrophobic filters and dedicated solutions Perform post PCR analysis in a separate area with a separate set of pipettors Clontech Laboratories Inc www clontech com Protocol No PT3024 1 40 Version No PR742227 Yeast Protocols Handbook VIII Analysis of Yeast Plasmid Inserts by PCR continued b Pipetting Because of the small volumes used in PCR experiments and the potential for tube to tube variation careful pipetting technique is extremely important Always be sure that no extra solution is on the outside
25. clontech com Protocol No PT3024 1 54 Version No PR742227 Yeast Protocols Handbook APPENDIX C Media Recipes continued X gal 20 mg ml in DMF Dissolve 5 bromo 4 chloro 3 indolyl B D galactopyranoside in N N dimethylformamide Store in the dark at 20 C 10X Dropout DO Solution Acombination ofa Minimal SD Base anda DO Supplementwill produce asynthetic defined minimal medium lacking oneormorespecificnutrients Thespecificnutrientsomitteddependsontheselection medium desired To prepare SD Leu Trp agar for example combine Leu Trp DO Supplement Cat No 630417 with Minimal SD Agar Base Cat No 630412 With this naming convention e g SD Leu Trpl if a nutrient is not indicated as missing it is assumed to be present in the medium Many of the commonly used DO Supplements can be purchased from Clontech Instructions for preparing the corresponding SD DO medium are printed on the products labels Alternatively make your own dropout supplement by combining the nutrients listed below at the concentrations indicated to prepare a 10X Dropout Solution A 10X Dropout Solution contains all but one or more of these nutrients Note that serine aspartic acid and glutamic acid are not included in this list because they make the medium too acidic and because yeast can synthesize these amino acids endogenously However if you wish to select for yeast strain AH109 using medium that lacks methionine i e on SD Met you sh
26. colony used for the inoculum will result in slow growth see Section II A 3 If you still have problems obtaining a healthy liquid culture streak a fresh working stock plate from the frozen glycerol stock and inoculate with a fresh colony Check the liquid medium to make sure it was made correctly If you suspect that the medium or carbon source stock solutions have been over autoclaved remake fresh solutions and either filter sterilize them or adjust the autoclave settings appropriately before autoclaving The addition of adenine hemisulfate toYPD in Steps E 3 and E 5 will enhance the growth of yeast strains that contain the ade2 101 mutation All of our Matchmaker host strains except EGY48 carry this mutation Check the concentration of the resuspended competent cells after Step E 11 using a hemocytometer If the cell concentration is lt 1 x 109 ml spin the cells down again at 1 000 x g for 5 min and resuspend them in a smaller volume of 1X TE LiAc buffer Occasionally there is a contaminant in the water that can affect transformation efficiency and or cell growth Prepare all reagents using sterile deionized distilled water such as Milli OTM filtered Confirm that your water purification system is functioning properly Clontech Laboratories Inc www clontech com Protocol No PT3024 1 22 Version No PR742227 Yeast Protocols Handbook VI a and p Galactosidase Assays A General Information Considerations e T
27. freeze thaw cycle at 20 C and vortex again to ensure complete lysis of the cells 6 If necessary samples can be stored frozen at 20 C If samples have been frozen vortex them again before using them 7 Pour the entire contents of the tube from Step 5 above onto a prespun CHROMA SPIN 1000 Column and purify the plasmid DNA according to the CHROMA SPIN User Manual Purified plasmid DNA will elute from the column If you do not use CHROMA SPIN Columns clean up the prep as follows a Bring the volume of the sample up to 200 ul inTE buffer pH 7 0 b Add 200 ul of phenol chloroform isoamyl alcohol 25 24 1 Vortex at highest speed for 5 min d Centrifuge at 14 000 rpm for 10 min e Transfer the aqueous upper phase to a fresh tube f Add 8 ul of 10 M ammonium acetate and 500 ul of 95 100 Ethanol h Place at 70 C or in a dry ice ethanol bath for 1 hr i Centrifuge at 14 000 rpm for 10 min j Discard supernatant and dry the pellet k Resuspend pellet in 20 ul of H O Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 35 Yeast Protocols Handbook Vil Working With Yeast Plasmids continued Note The amount of plasmid DNA recovered is small relative to the contaminating genomic DNA therefore it cannot be measured by A or seen on an agarose gel C Transforming E coli with Yeast Plasmids We recommend using electroporation Section C 1 when transforming E col
28. inhibitor solution Appendix D A PMSF stock solution Appendix D A Cracking buffer stock solution Appendix D A Cracking buffer complete Appendix D A Note Unless otherwise stated keep protein samples on ice 1 5 6 Prepare complete cracking buffer Appendix D A and prewarm it to 60 C Because the PMSF degrades quickly prepare only the amount of cracking buffer you will need immediately Use 100 ul of cracking buffer per 7 5 units of cells For example for 33 total OD units of cells use 0 44 ml of cracking buffer Quickly thaw cell pellets by separately resuspending each one in the prewarmed cracking buffer If cell pellets are not immediately thawed by the prewarmed cracking buffer place the tubes briefly at 60 C to hasten melting To avoid risk of proteolysis do not leave them longer than 2 min at 60 C Because the initial excess PMSF in the cracking buffer degrades quickly add an additional aliquot of the 100X PMSF stock solution to the samples after 15 min and approximately every 7 min thereafter until Step 9 when they are placed on dry ice or are safely stored at 70 C or colder Use 1 ul of 100X PMSF per 100 ul of cracking buffer Transfer each cell suspension to a 1 5 ml screw cap microcentrifuge tube containing 80 ul of glass beads per 7 5 units of cells Note The volume of the glass beads can be measured using a graduated 1 5 ml microcentrifuge tube Heat samples at 70
29. interest Refer to Appendix E for information on yeast plasmid transformation selection markers Note These yeast clones are referred to as Cyh segregants Store them on the appropriate SD selection plates wrapped in Parafilm at 4 C for up to two weeks C Yeast Mating Yeast mating is a convenient method of introducing two different plasmids into the same host cells and in some applications can be used as a convenient alternative to yeast cotransformations Bendixen et al 1994 Harper et al 1993 Finley amp Brent 1994 See Guthrie amp Fink 1991 or Pringle et a 1997 for information on the biology of yeast mating The following small scale protocol works well for creating diploids by yeast mating If you wish to screen a Pretransformed Matchmaker Libary using yeast mating please refer to the User Manual provided with those libraries for an optimized library scale mating protocol 1 Preparation for yeast mating a If you have not done so already generate an appropriate yeast strain containing the plasmid of interest b Transform the chosen mating partner separately with the plasmids you wish to test in combination with the plasmid of interest Be sure to include transformations with the appropriate negative and positive control plasmids if applicable Clontech Laboratories Inc www clontech com Protocol No PT3024 1 44 Version No PR742227 Yeast Protocols Handbook IX Additional Useful Protocols continued c
30. into fresh medium to produce an OD 0 2 0 3 Incubate at 30 C for 3 5 hr with shaking 230 250 rpm This will with most strains produce a culture with an OD 0 4 0 6 Note Generally YPD orYPDA may be used in this incubation Because of the shorter incubation time plasmid loss will not be significant However do not use YPD if you want to induce protein expression from the yeast GAL7 promoter of a LexA system plasmid e g pB42AD or pGilda YPD contains glucose which represses transcription from the GAL7 promoter Clontech Laboratories Inc www clontech com Protocol No PT3024 1 10 Version No PR742227 Yeast Protocols Handbook 11 Culturing and Handling Yeast continued B Growth Selection for Transformation Markers and Reporter Gene Expression Most yeast cloning vectors and control plasmids including those provided in our Matchmaker Systems carry at least one nutritional marker to allow for selection of yeast transformants plated on SD minimal medium lacking that specific nutrient Furthermore if you are cotransforming yeast with two or more different plasmids bearing different nutritional markers the plasmids can be independently selected Thus the SD selection medium you choose for plating transformants depends generally on the purpose of the selection Specific factors to consider in choosing the appropriate SD selection medium are the plasmid s used and whether you are selecting for one or more plasmids w
31. l Klein R amp Hiebsch R Ed CRC Press Boca Raton Fl page 61 Clontech Laboratories Inc www clontech com Protocol No PT3024 1 48 Version No PR742227 XI Matchmaker and Related Products Yeast Protocols Handbook For the latest and most complete listing of all Clontech products please visit www clontech com General reagents for work with yeast Yeastmaker Yeast Transformation System Yeastmaker Carrier DNA Yeastmaker Yeast Plasmid Isolation Kit YPD Medium YPD Agar Medium Minimal SD Base contains glucose Minimal SD Agar Base contains glucose Minimal SD Base Gal Raf contains galactose amp raffinose Minimal SD Agar Base Gal Raf contains galactose amp raffinose Dropout DO Supplements for use with any SD Base GAL4 based one and two hybrid systems and related products Mammalian Matchmaker Two Hybrid Assay Kit Matchmaker One Hybrid System Matchmaker Two Hybrid System 3 Matchmaker Co IP Kit pCMV Myc amp pCMV HA Vector Set Matchmaker cDNA amp Genomic Libraries Matchmaker Pretransformed Libraries Matchmaker AD LD Insert Screening Amplimer Set GAL4 AD Monoclonal Antibody GAL4 DNA BD Monoclonal Antibody pGBKT7 DNA BD Vector pGADT7 AD Vector pLP GBKT7 DNA BD Creator Acceptor Vector pLP GADT7 AD Creator Acceptor Vector pBridge Three Hybrid Vector General cloning reagents QUICK Clone cDNA Genomic DNA E coli KC8 Electrocompetent Cells E coliKC8 Chemically C
32. lon cont Plasmid Informati APPENDIX E paljipow Spiuuse d y u p qu s p spiuuse d ay JO S ARPAH P 40910012 L EVAd pue L Lqid eg yxeqd L GIAIVTd GiNYTd L avzrdd e 1661 ye 39 4 1 670 ZL 272 97 due Lgl 661 29 iuonqeAN III pu H v6 daj L zsvd ui 06572 ggd uunui Z HZ Y Y 1 VAd 87 9 voce zn AWE 661 29 iuonqeAN III pu H v9 d l 6LaDd 0822 ggd uunu HZ r1V9 VAd 410 OL ZL Z a fe 0131 1OVd u 16661 SPIE 1 III pu H 0 OL n 7 80278 usBique e648 OVAS 2 2 VIVD L Lad 1661 29 u u5 90 ZL EL ZL dwe 27047 4 ul 661 Spa 1 III pU H O GL ney 07778 YaBrue abe OVAS HZ Laid v LLHS 076 Gv 7079 dwe ESIH 7661 8 19 siuu9 oD III pu H g SIH auaB uolsn p7VD VxXeT HZ Vx91 sod Vx 1d 270 6 0 6 Z3 dwe ESIH Vx 1d u Peg661 P joe III pu H 901 SIH 87999 ujue uetunH HZ Vx91 uie1 Vx 1d z o s Z 9 dwe ESIH 661 2 19 1yonqem III Pu H Ul SIH vx rd ul ead uunuu HZ vx q t o 4870 670 ZZ 9 due Idy 1 290 u 86661 29 jane III pu H 0 67 d l 087999 ule uewnyH Z HZ Y V L SIAV1d 9 0 870 Z v dwe dul 61800 u 96661 e 19 j ueg III pU H 09 d l 27 999 urusel ueuunH HZ SINV Td 490
33. of culture The concentration factor from Step D 7 is 5 However it may be necessary to try several dilutions of cells at this step hence different concentration factors to remain within the linear range of the assay E Liquid Culture Assay Using CPRG as Substrate Reagents and Materials Required Appropriate liquid medium Appendix C A 50 ml culture tubes Buffer 1 Appendix D Buffer 2 Appendix D CPRG chlorophenol red B D galactopyranoside Roche Applied Science Cat No 10884308001 3 mM ZnCl Filter sterilized to preserve for 3 months Liquid nitrogen 1 Prepare 5 ml overnight cultures in liquid SD medium as described in Chapter Ill A 3 Usethe SD selection medium appropriate for your system and plasmids Note Be sure to use SD medium that will maintain selection on the plasmids used 2 Vortex the overnight culture tube for 0 5 1 min to disperse cell clumps Immediately Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Vi Yeast Protocols Handbook a and 6 Galactosidase Assays continued N transfer 2 ml of the overnight culture to 8 ml of YPD except for LexA System Note For the LexA System use the appropriate SD Gal Raff induction medium for the strains being assayed 3 Incubate fresh culture at 30 C for 3 5 hr with shaking 230 250 rpm until the cells are in mid log phase OD of 1 ml 0 5 0 8 Record the exact OD when you harvest the cells
34. of the pipette tip before transfer When adding solution to a tube immerse the tip into the reaction mixture deliver the solution and rinse the pipette tip by pipetting up and down several times c Use a Master Mix To reduce tube to tube variation use a master mix whenever you set up multiple PCR reactions If you wish include the primers in the master mix also If you are setting up several sets of parallel samples assemble multiple master mixes e g each with a different set of primers The master mix should be thoroughly mixed before use i e vortexed without bubbling d Always include positive and negative controls i e H O instead of DNA template Positive controls are provided with all of Clontech s Insert Screening Amplimer Sets Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 41 Yeast Protocols Handbook IX Additional Useful Protocols A Yeast Colony Hybridization Yeast colony hybridization is an efficient way to screen a large collection of library transformants for the presence of an abundantcDNA insert Duplicate colonies bearing the same library plasmid can then be eliminated from further analysis We have had success with this modification of the classic protocol of Grunstein and Hogness 1975 Kaiser et al 1994 Ausubel et al 1994 In this procedure colonies are directly lifted onto a nylon membrane B glucuronidase is used to break cell walls Reagents and
35. provides a Advantage 2 Polymerase Mix which includes TagStart Antibody a 10X Advantage 2 PCR Buffer 50X dNTP a positive control template a mix of positive control primers and a complete User Manual Clontech offers PCR primers designed to amplify inserts cloned into Matchmaker Two Hybrid System vectors The insert screening amplimers hybridize to sequences flanking the multiple cloning site MCS of the respective vectors If you purchase Matchmaker LD Insert Screening Amplimers we recommend that you use the LD PCR protocol that accompanies that product However LD Insert Screening Amplimers can also be used in conventional PCR using a single DNA polymerase to amplify inserts up to 3 kb e g Ausubel et al 1995 Chapters 15 1 amp 15 3 Matchmaker AD LD Insert Screening Amplimers Cat No 630433 are for amplifying inserts in the GAL4 AD cloning vectors pGAD10 pGAD424 pGAD GL pGAD GH and pACT2 e Matchmaker pB42AD LD Insert Screening Amplimers Cat No 9108 1 are for amplifying inserts in the LexA system AD cloning vector pB42AD e Matchmaker DNA BDVector Insert Screening Amplimers Cat No 5417 1 are for conventional PCR amplification of inserts in the GAL4 DNA BD cloning vectors pGBT9 pGBKT7 pAS2 and pAS2 1 MatchmakerLexA DNA BD Insert Screening Amplimers Cat No 9109 1 are for conventional PCR amplification of inserts in pLexA and pGilda B Tips For Successful PCR of Yeast Pl
36. purified and contain lt 0 01 glucose e Optional For 3 AT containing medium add the appropriate amount of 1 M 3 AT stock solution and swirl to mix well The concentration of 3 AT used in the medium depends on the yeast strain and to some extent on the presence of transforming plasmid s See your system specific User Manual for further information Notes e 3 AT is heat labile and will be destroyed if added to medium hotter than 55 C 3 AT a competitive inhibitor of the yeast H S3 protein His3p is used to inhibit low levels of His3p expressed in a leaky manner in some reporter strains Fields 1993 Durfee et al 1993 Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 53 Yeast Protocols Handbook APPENDIX C Media Recipes continued e Optional For cycloheximide containing medium add the appropriate amount of 1 mg ml cycloheximide stock solution and swirl to mix well The concentration of cycloheximide used in the medium depends on the yeast strain See your system specific User Manual for further information Notes e Cycloheximide is heat labile and will be destroyed if added to medium hotter than 55 C e Cycloheximide containing medium is used for selection of yeast strains such asY190 and CG 1945 carrying the cyh 2 allele e Optional If you wish to add excess adenine to SD medium add 15 ml of 0 2 adenine hemisulfate solution per liter of medium Pour plates and
37. to thoroughly disperse the cells Notes e Liquid cultures will grow slower than expected if clumps are present in the inoculum cells in the interior of the clumps do not have access to the nutrients in the medium e If you are inoculating a volume greater than 1 ml it is easier to disperse the clumps if the colonies are first placed in 1 ml of medium in a microcentrifuge tube vortexed and then transferred to the desired volume e When growing overnight cultures of yeast transformants use the appropriate SD minimal medium to keep selective pressure on extrachromosomal plasmid s The growth inYPD of yeast strains carrying the ade2 107 mutation will be enhanced by adding adenine hemisulfate 0 003 final concentration to the medium Appendix C A All of the host strains except EGY48 used in the Matchmaker Systems carry this auxotrophic mutation The growth of transformed PJ69 2A cells in SD Trp may also be enhanced by adding excess adenine to the medium Appendix C A Incubate at 30 C for 16 18 hr with shaking at 230 270 rpm With most strains this will yield a stationary phase culture OD gt 1 5 Note Different yeast strains grow at different rates Growth rates may also be affected by the presence of fusion proteins in certain transformants In addition the doubling time of most strains growing in SD minimal medium is twice as long as in YPD If you need a mid log phase culture transfer enough of the overnight culture
38. 0 20 min 2 Resuspend each cell pellet in 100 ul of ice cold TCA buffer per 75 OD units of cells For example for 33 total OD units of cells use 0 44 ml of TCA buffer Place tubes on ice 3 Transfer each cell suspension to a 1 5 ml screw cap microcentrifuge tube containing glass beads and ice cold 20 TCA Use 100 ul of glass beads and 100 ul of ice cold 20 TCA per 7 5 OD units of cells Note The volume of the glass beads can be measured using a graduated 1 5 ml microcentrifuge tube 4 To disrupt cells place tubes in a Bead Beater and set speed at highest setting Bead beat the cells for 2 X 30 sec placing tubes on ice for 30 sec in between the two bead beatings Place tubes on ice Note If you do not have access to a Bead Beater you can vortex the tubes vigorously at 4 C for 10 min alternatively you can vortex at room temperature for shorter periods of 1 min each at least 4 times placing tubes on ice for 30 sec in between each vortexing Place tubes on ice 5 Transfer the supernatant above the settled glass beads to fresh 1 5 ml screw cap tubes and place tubes on ice This is the first cell extract Note The glass beads settle quickly so there is no need to centrifuge tubes at this point 6 Wash the glass beads as follows a Add 500 ul of an ice cold 1 1 mixture of 20 TCA and TCA buffer b Place tubes in Beat Beater and beat for another 30 sec at the highest setting Alternatively vortex for 5 min at 4 C or
39. 0 5 1 min to thaw 14 Repeat freeze thaw cycle Steps 12 amp 13 once to ensure that cells have been cracked open 15 Warm to room temperature enough reaction buffer for the entire experiment 16 Set up a blank tube with 25 ul of Z buffer 17 Optional If you wish to obtain absolute as well as relative data set up a series of B galactosidase standard tubes containing 0 0005 0 001 0 003 0 010 and 0 020 unit of B galactosidase in 25 ul of Z buffer 18 Place 20 30 ul of each cell lysate in a separate sample tube or into wells of an opaque 96 well flat bottom microtiter plate suitable for plate luminometers If you are using a sample tube the tube should hold at least 0 5 ml Note The amount of yeast extract required may vary depending upon the level of B gal expression and the detection device used Use 10 30 ul of extract for positive controls and 20 30 ul for experimental samples with potentially low levels of enzyme activity It is important to vary the amount of extract to keep the signal within the linear range of the assay 19 Add 200 ul of Galacton Star reaction mixture to each sample tube or well and mix Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook VI a and B Galactosidase Assays continued gently 20 Incubate at room temperature 20 25 C for 60 min Note Light signals produced during this incubation are stable for gt 1 hr therefore
40. 0410 are available in powder form from Clontech Our YPD Medium is a blend of peptone yeast extract and dextrose in optimal proportions for growth of most strains of Saccharomyces cerevisiae See Chapter XI for ordering information If you purchase Clontech sYPD media prepare the medium according to the instructions provided Alternatively prepare your own YPD mixture as follows 20 g L Difco peptone 10 g L Yeast extract 20 g L Agar for plates only e Optional For adenine supplementedYPD YPDA add 15 ml of a0 2 adenine hemisulfate solution per liter of medium final concentration is 0 003 in addition to the trace amount of Ade that is naturally present in YPD Adenine hemisulfate tolerates autoclaving Add H O to 950 ml Adjust the pH to 6 5 if necessary then autoclave Allow medium to cool to 55 C and then add dextrose glucose to 2 50 ml of a sterile 40 stock solution Adjust the final volume to 1 L if necessary Note If you add the sugar solution before autoclaving autoclave at 121 C for 15 min autoclaving at a higher temperature for a longer period of time or repeatedly may cause the sugar solution to darken and will decrease the performance of the medium Note thatYPD from Clontech already contains glucose e Optional Forkanamycin containing medium prepareYPD orYPDA as above After autoclaved medium has cooled to 55 C add 0 2 0 3 ml of 50 mg ml kanamycin final concentration 10 15 mg L SD medium
41. 604 1 K1605 1 630303 and LexA Two Hybrid System Cat No K1609 1 are complete kits for identifying and investigating protein protein interactions in vivo using the yeast two hybrid assay The Matchmaker One Hybrid System Cat No K1603 1 provides the basic tools for identifying novel proteins in vivo that bind to a target DNA sequence such as a cis acting regulatory element Matchmaker Two Hybrid Systems are compatible with our pBridgeThree Hybrid Vector Cat No 630404 for the investigation of tertiary protein complexes The Matchmaker Libraries are constructed in vectors that express inserts as fusions to a transcriptional activation domain and are thus a convenient resource for researchers wishing to screen a library using the one or two hybrid assays Pretransformed Matchmaker Libraries provide an even greater level of convenience for those wishing to perform a two hybrid library screening without using large or library scale yeast transformations Clontech offers an extensive line of kits and reagents that support and complement the Matchmaker Systems and Libraries The Yeastmaker Yeast Transformation Kit Cat No 630439 includes all the necessary reagents and protocols for efficient transformation using the lithium acetate method Also available from Clontech a selection of GAL4 DNA binding domain DNA BD and activation domain AD hybrid cloning vectors the pGilda Vector for use with LexA based two hybrid systems monoclonal antibodie
42. 61 8 19 ON l OUX I YOIF Lg SIH ean JO 104u09 1 9pun ESIH SIH Gd q qx as quondu s q e10 39A SOUS FY z is uo uon l s nsou5eiq SGIINSV1d TOHINO 79 1 4 WALSAS HANV NH LV N MA 414V1 www clontech com Clontech Laboratories Inc Protocol No PT3024 1 Version No PR742227 63 Yeast Protocols Handbook ion Yeast Host Strain Informat APPENDIX F jonsjsuod s nod l Z39V ZTV u1 YUM uoneuiquioo 1 Aq sem 6 1osino 1d y u B 01 C DP L 7661 8 19 nol 93 ILAH JO ARBAH P L S GHEL DD s esq JaxeWIYO e A D uuioJjsue119id S LA Ld p UH 3SAS 5141 u SJOxJELU uolo s Se p sn 31e 181 s u D sadAjouaud 1194 pue s u B yod UO uonguuioJul 10 A QEL S q S U B 1 uod 1 y JO S19 OWOJd v UO uoneuuoJul 104 JAIdeEUD 29S G 61 5 pUe 9 4H 24 u euonounjuou si BUBB ZSAT 89ul uone1nui lqnop e si Z L e cnaj suon f p Ile aie suonen 08 26 pue pjeb situ Idi SUL e 228 4 Wwe 1 T3W gvun 2234 2199 210 p us lqndun z loH L13W LTAW SIH YY IVv9 S L7V9 ZSAT VOS P vp eB 19661 Je s uuer znal idi Z98 2307 ESIH 1002 6914 ZG E84AN 211 Zn l L06 L di ELVIN HZ 60LHV 1130 Z3av ZTV ZTV SIH E
43. A lQET EAB jou III pu H 69 n 1 av S y s neiqi7 NN T qv5d 20 40 LZ dwe 2131 666 8 19 Isjay ULA uolssiuiqns ul III pu H 61 n 1 qv 99990 q s neq NWN H qv5d 0 6 G dwe 2 137 E GGL Je Ja joueg 9 0 III pu H 9 9 n 1 av 9 2172 2 1 2 0 6 4 dwe 2 137 6661 Je j ueg 88161 III pu H 99 n 1 av 7yo s nesq N N oLav d 9 4610 970 4112 176 vH dwe 1441 6661 8 19 SUNAD 19668 III pu H sg daj Zyg Joyeanoe 91p19e HZ yx97 avzvad 70 60 ZZ 91 ZsHAD dwe Pq 6661 s dieH L6v0EN III pu H v8 daj qg vNq z7y 2 2 VIVD oL zSwd S d 13 80 Z vH dwe 2 1 s ueiqi N N 3 7661 8 1 11 66862 III pu H 8 n 1 av 8 8 b7q75 2 2 VIVD zLowd si d 3 97 GO E 012 vH dwe 7947 16661 19 junq Jou I 4097 497 ney av 85775 s nesq WIN Lovd u ulpu ON uolss oy qJ 5919 3 u 44 wnIp N GS 15 1O J99A s u s qg yuegu S nsouBelq z s uo uon o l s NI NOTO WALSAS AIHAAH OML H V NH LV N A 318V1 www clontech com Clontech Laboratories Inc Protocol No PT3024 1 Version No PR742227 61 Yeast Protocols Handbook nued i
44. ADE2 and LEU2 reporter genes are under control of artificial promoter constructs comprised of aTATA and UAS or operator sequence derived from another gene Table 1 In some cases the TATA sequence and the UAS are derived from different genes indeed the LexA operator is a cis acting regulatory element derived from E coli For GAL4 based systems either a native GAL UAS or a synthetic UAS consensus sequence Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 5 Yeast Protocols Handbook II Introduction to Yeast Promoters continued TABLE 1 YEAST PROMOTER CONSTRUCTS USED TO REGULATE REPORTER GENE EXPRESSION IN MATCHMAKER PLASMIDS AND HOST STRAINS Plasmid or Reporter Origin of UAS Origin of Expression level host strain gene UAS regulated by TATA sequence Induced uninduced CG 1945 lacZ UAS 17 mer x3 GAL4 CYC1 lovv HIS3 GAL1 GAL4 GAL1 high slightlyleaky HF7c lacZ UAS b6 17 mer x3 GAL4 CYC1 lovv HIS3 GAL1 GAL4 GAL1 high tight Y190 lacZ GAL1 GAL4 GAL1 high HIS3 GAL1 GAL4 HIS3 TC TR high leaky Y187 lacZ4 GAL1 GAL4 GAL1 high SFY526 lacZ GAL1 GAL4 GAL1 high PJ69 2A HIS3 GAL1 GAL4 GAL1 high tight ADE2 GAL2 GAL4 GAL2 high tight AH109 HIS3 GAL1 GAL4 GAL1 high tight ADE2 GAL2 GAL4 GAL2 high tight lacZ MEL1 GAL4 MEL1 low EGY48 LEU2 LexA opus LexA LEU2 high p8op lacZ lacZ LexA LexA GALT high pHISi HIS3 none n a HIS3 TC TR n a leaky pHI
45. AL2 MEL1 UASs and the 17 mer 6 Figure 2 Urea SDS protein extraction method 14 Figure 3 TCA protein extraction method 16 Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diagnostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without written approval of Clontech Laboratories Inc Practice of the two hybrid system is covered by U S Patent Nos 5 283 173 5 468 614 and 5 667 973 assigned to the Research Foundation of the State University of NewYork Purchase of any Clontech two hybrid reagent does not imply or convey a license to practice the two hybrid system covered by these patents Commercial entities purchasing these reagents must obtain a license from the Research Foundation of the State University of NewYork before using them Clontech is required by its licensing agreement to submit a report of all purchasers of two hybrid reagents to SUNY Stony Brook Please contact the Office of Technology Licensing amp Industry Relations at SUNY Stony Brook for license information Tel 631 632 9009 Fax 631 632 1505 NOTICE TO PURCHASER LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding pate
46. D with several colonies 2 3 mm in diameter Note For host strains previously transformed with another autonomously replicating plasmid use the appropriate SD selection medium to maintain the plasmid Appendix E Vortex vigorously for 5 min to disperse any clumps Transfer this into a flask containing 50 ml of YPD or the appropriate SD medium Incubate at 30 C for 16 18 hr with shaking at 250 rpm to stationary phase OD gt 1 5 Transfer 30 ml of overnight culture to a flask containing 300 ml of YPD Check the OD of the diluted culture and if necessary add more of the overnight culture to bring the OD up to 0 2 0 3 Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook V Yeast Transformation Procedures continued 6 Incubate at 30 C for 3 hr with shaking 230 rpm At this point the OD should be 0 4 0 6 Note If the OD is lt 0 4 something is wrong with the culture see Troubleshooting Section F6 7 Place cells in 50 ml tubes and centrifuge at 1 000 x g for 5 min at room temperature 20 21 C 8 Discard the supernatants and thoroughly resuspend the cell pellets in sterileTE or distilled H O Pool the cells into one tube final volume 25 50 ml 9 Centrifuge at 1 000 x g for 5 min at room temperature 10 Decant the supernatant 11 Resuspend the cell pellet in 1 5 ml of freshly prepared sterile 1X TE 1X 12 Add 0 1 ug of plasmid DNA a
47. Flick amp Johnston 1990 Johnston et al 1994 or gal4A GAL80 gal80 538 Gal Deficient in regulation of galactose metabolizing genes GAL genes are constitutively expressed CYH 2 cyh 2 Cyh Resistant to cycloheximide TABLE V REPORTER GENES AND THEIR PHENOTYPES Reporter Gene Positive Negative Gene Description Phenotype Phenotype lacZ Encodes 8 galactosidase LacZ LacZ e Blue colony e White colony e B gal activity above e Undetectable or background background level of B gal activity HIS3 Confers His prototrophy His His e Grows on SD His Does not grow on 50 159 LEU2 Confers Leu prototrophy Leu Leu e Grows on SD Leu Does not grovv on SD Leu ADE2 Confers Ade prototrophy Ade Ade Grows on SD Ade Does not grow on 50 e Pink or red colony color when grown on medium such as YPD low in Ade a Relative levels of background expression and reporter gene induction are dependent on the promoter constructs controlling them See Chapter l for information on the promoters gt 5 60 mM 3 AT may be required to suppress leaky H S3 expression in certain host strains and transformants and to obtain an accurate His phenotype Clontech Laboratories Inc www clontech com Protocol No PT3024 1 52 Version No PR742227 Yeast Protocols Handbook APPENDIX C Media Recipes A YEAST MEDIA YPD medium YPD Medium Cat No 630409 and YPD Agar Medium Cat No 63
48. GAL4 DNA BD mAb 0 5 ug ml or GAL4 AD mAb 0 4 ug ml using 1 ml of diluted mAb per 10 cm of blot followed by HRP conjugated polyclonal Goat Anti Mouse IgG Jackson Immunological Research diluted 1 15 000inTBST Signals were detected using a chemiluminescent detection assay and a 2 5 min exposure of x ray film Signal intensities were compared to that of known amounts of purified GAL4 DNA BD a a 1 147 or GAL4 AD a a 768 881 The truncated ADH7 promoter in pACT2 is adjacent to a section of pBR322 which acts as a transcriptional enhancer in yeast d Data obtained using EGY48 p8o0p lacZ transformed with pGilda and grown in the presence of glucose or galactose respectively April 1997 Clontechniques no data available for pB42AD Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 Yeast Protocols Handbook III Culturing and Handling Yeast For additional information on yeast we recommend Guthrie and Fink 1991 Guide to Yeast Genetics and Molecular Biology Cat No V2010 1 A Yeast Strain Maintenance Recovery from Frozen Stocks and Routine Culturing 1 Long term storage e Yeast strains can be stored indefinitely inYPD medium with 25 glycerol at 70 C For storage gt 1 year the temperature must be maintained below 55 C e Transformed yeast strains are best stored in the appropriate SD dropout medium to keep selective pressure on the plasmid See Appendix C A for recipes and Append
49. Length and G C content In general primers should have a T of at least 70 C to achieve Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 39 Yeast Protocols Handbook VIII Analysis of Yeast Plasmid Inserts by PCR continued optimal results in a two step cycling program with a 68 C annealing extension step Therefore whenever possible primers should be at least 22 nucleotides nt long 25 30 mers are preferred and should have a GC content of 45 60 3 Thermostable polymerase The Advantage 2 Polymerase Mixes are designed for LD PCR i e they contain both primary and proofreading polymerases to permit amplification of virtually any insert regardless of size If you do not use an Advantage Polymerase Mix you will need to prepare your own polymerase mix from commercially available LD PCR licensed DNA polymerases such as Taq or AmpliTaq We also strongly recommend that you include TaqStart Antibody in the polymerase mix for automatic hot start see Section B 4 below TaqStart Antibody is premixed in the Advantage 2 Polymerase Mix 4 Use of antibody mediated or conventional hot start To minimize nonspecific amplification we strongly recommend that you perform hot start PCR There are several methods available for hotstart PCR including those using wax beads Chou et al 1992 or a manual hot start D Aquila et al 1991 TaqStart Antibody Cat No 639250 639251 provides an au
50. MSF phenylmethyl sulfonyl fluoride stock solution 100X Dissolve 0 1742 g PMSF Sigma Cat No P7626 in 10 ml isopropanol Wrap tube in foil and store at room temperature PMSF primarily inhibits serine proteases Although this is a 100X stock solution the final concentration of PMSF is greater than 1X in some mixtures i e PMSF is used in excess Caution PMSF is hazardous Wear gloves Handle with care and read label precautions e Glass Beads 425 600 um Sigma Cat No G 8772 For Urea SDS Protein Extraction Method e Cracking buffer stock solution To prepare 100 ml Urea 8 M 48 g SDS 5 wiv 5g Tris HCI pH6 8 40 mM 4 ml of a1 M stock solution EDTA 0 1 mM 20 ul of a 0 5 M stock solution Bromophenol blue 0 4 mg ml 40mg Deionized H O To a final volume of 100 ml EDTA primarily inhibits metalloproteases Cracking buffer complete The following recipe is sufficient for one protein extract Scale up recipe as required Prepare only the volume you need just before use Because PMSF has a short half life 7 min in aqueous solutions you may need to add additional aliquots of PMSF during the course of the procedure The initial excess PMSF in the Cracking buffer quickly degrades To prepare 1 13 ml of complete Cracking buffer Cracking buffer stock solution 1 ml recipe above B mercaptoethanol 10 ul Protease inhibitor solution 70 ul prechilled recipe above PMSF 50 ul of 100X stock solution Protocol N
51. R amplification of up to 35 kb DNA with high fidelity and high yield from A bacteriophage templates Proc Natl Acad Sci USA 91 2216 2220 Beier D R amp Young E T 1982 Characterization of a regulatory region upstream of the ADR2 locus of S cerevisiae Nature 300 724 728 Bendixen C Gangloff S amp Rothstein R 1994 A yeast mating selection scheme for detection of protein protein interactions Nucleic Acids Res 22 1778 1779 Bolivar F amp Backman K 1979 Plasmids of Escherichia coli as cloning vectors Methods Enzymol 68 245 267 Breeden L amp Nasmyth K 1985 Regulation of the Yeast HO Gene Cold Spring Harbor Symposium Quant Biol 50 643 650 Campbell K S Buder A amp Deuschle U 1995 Interactions between the amino terminal domain of p56 and cytoplasmic domains of CD4 and CD8 alpha in yeast Eur J Immunol 25 2408 2412 Campbell K S Buder A Deuschle U 1995 Interaction of p56 with CD4 in the yeast two hybrid system Ann NY Acad Sci 766 89 92 Cheng S Fockler C Barnes W M amp Higuchi R 1994 Effective amplification of long targets from cloned inserts and human genomic DNA Proc Natl Acad USA 91 5695 5699 Chien C T Bartel P L Sternglanz R amp Fields S 1991 The two hybrid system A method to identify and clone genes for proteins that interact with a protein of interest Proc Nat Acad Sci USA 88 9578 9582 Chou O Russell M Birch D R
52. Si 1 HIS3 none n a HIS3 TC TR n a leaky pLacZi lacZ none n a CYC1 n a tight 3 See Appendices E 8 F for references b When induced by a positive two hybrid interaction leaky and tight refer to expression levels in the absence of induction Conserved 17 bp palindromic sequence to which the GAL4 protein binds Guthrie amp Fink 1991 d Y187 probably contains two copies of the lacZ gene judging by the strength of the signal in this strain and in the strains from which it was derived Durfee et a 1993 Harper et al 1993 This is the minimal TATA region of the GAL7 promoter it does not include the GAL 7 UAS and therefore is not responsive to regulation by GAL4 protein f The Matchmaker One Hybrid System vectors do not contain a UAS because they are used to experimentally test target elements inserted upstream of the minimal promoter for their ability to bind specific transcriptional activators In the absence of inserted target elements reporter gene expression is not induced however expression levels may be leaky depending on the nature of the minimal promoter used in that vector GAL1 bs1 TAGAAGCCGCCGAGCGG GAL1 UAS GAL1 bs2 GACAGCCCTCCGAAGGA GAL1 bs3 GACTCTCCTCCGTGCGT MEL1 UAS CGGCCATATGTCTTCCG GAL1 bs4 CGCACTGCTCCGAACAA GAL2 bsl CGGAAAGCTTCCTTCCG GAL2 bs2 CGGCGGTCTTTCGTCCG GAL UAS GAL2 bs3 CGGAGATATCTGCGCCG UAS 17 CGGAAGACTCTCCTCCG GAL2 bs4 CGGGGCGGATCACTCCG GAL2 bs5
53. Sons Inc Chapters 20 0 and 20 1 Golemis E A Gyuris J amp Brent R 1994 Interaction trap two hybrid systems to identify interacting proteins In Current Protocols in Molecular Biology John Wiley amp Sons Inc Ch 13 14 Guarente L 1993 Strategies for the identification of interacting proteins Proc Natl Acad Sci USA 90 1639 1641 Gyuris J Golemis E Chertkov H amp Brent R 1993 Cdi1 ahuman G1 and S phase protein phosphatase that associates with Cdk2 Cell 75 791 803 Luban J amp Goff S P 1995 The yeast two hybrid system for studying protein protein interactions Curr Opinion in Biotechnol 6 59 64 Mendelsohn A R amp Brent R 1994 Biotechnology applications of interaction traps two hybrid systems Curr Opinion in Biotechnology 5 482 486 Other References Aho S Arffman A Pummi T 8 Uitto J 1997 A novel reporter gene MEL7 for the yeast two hybrid system Anal Biochem 253 270 272 Alexandre C Grueneberg D A amp Gilman M Z 1993 Studying Heterologous Transcription Factors inYeast METHODS A Companion to Methods in Enzymology 5 147 155 Ammerer G 1983 Expression of Genes in Yeast Using the ADC Promoter Methods in Enzymology 101 192 201 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A Struhl K 1994 Current Protocols in Molecular Biology John Wiley amp Sons Inc Vol 1 Chap 5 Barnes W M 1994 PC
54. Yeast Protocols Handbook FOR RESEARCH USE ONLY Visitiour website 7 for more details 86 click here PT3024 1 PR742227 Published 11 February 2008 Yeast Protocols Handbook Table of Contents VI VII VIII X XI Introduction Introduction to Yeast Promoters Culturing and Handling Yeast Preparation of Yeast Protein Extracts A General Information B Preparation of Yeast Cultures for Protein Extraction C Preparation of Protein Extracts Urea SDS Method D Preparation of Protein Extracts TCA Method E Troubleshooting Yeast Transformation Procedures General Information Reagents and Materials Required Tips for a Successful Transformation Integrating Plasmids into the Yeast Genome Small scale LiAc Yeast Transformation Procedure Troubleshooting Yeast Transformation g mm omp and b Galactosidase Assays General Information In vivo Plate Assay Using X gal in the Medium Colony lift Filter Assay Liquid Culture Assay Using ONPG as Substrate Liquid Culture Assay Using CPRG as Substrate Liquid Culture Assay Using a Chemiluminescent Substrate a Gal Quantitative Assay ommppmp VVorking vvith Yeast Plasmids A General nformation B Plasmid Isolation From Yeast C Transforming E coli with Yeast Plasmids Analysis of Yeast Plasmid Inserts by PCR A General Information B Tips for Successful PCR of Yeast Plasmid Templates Additional Useful Protocols A Yeast Colony Hybr
55. allow medium to harden at room temperature Store plates inverted in a plastic sleeve at 4 C e SD Gal Raf X gal plates Prepare SD medium as described above except use 725 ml of H O and do not adjust the pH Autoclave and cool to 55 C Then add Final concentration To prepare 1 L of medium Galactose 2 50 ml of 40 stock Raffinose 1 25 ml of 40 stock 10X BU salts 1X 100 ml of 10X stock X Gal 80 mg L 4 ml of 20 mg ml Pour plates and allow medium to harden at room temperature Store plates inverted in a plastic sleeve in the dark at 4 C for up to two months Adjust final volume to 1L if necessary Notes Galactose must be highly purified and contain lt 0 01 glucose e Ifthe medium is too hot i e gt 55 C when the salt solution is added the salts will precipitate Also X Gal is heat labile and will be destroyed if added to hot medium BU salts must be included in the medium to adjust the pH to 7 which is closer to the optimal pH for B galactosidase activity and to provide the phosphate necessary for the B gal assay to work e As the plates age salt crystals will form in the medium These do not affect the performance of the medium or the results of the B galactosidase assay Ifyou are assaying for expression of a lacZ reporter gene in a system that requires expression of a protein from an intact yeast GAL7 promoter such as in the Matchmaker LexA Two Hybrid System you must use 2 galactose 1 raffin
56. amp Struhl 1995 is not regulated by the native GCN4 binding UAS the GAL 1 UAS or artificial UAS constructs Mahadevan amp Struhl 1990 Hope amp Struhl 1986 The H S3 reporter gene in yeast strainY190 is unusual among the GAL4 two hybrid reporter gene Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 7 Yeast Protocols Handbook ll Introduction to Yeast Promoters continued constructs in that it is under the control of the GAL7 UAS and a minimal promoter containing both HIS3 TATA boxes Flick amp Johnston 1990 The result is high level expression due to the GAL7 UAS when induced by a positive two hybrid interaction this construct also exhibits a significant level of constitutive leaky expression due to the H S3TC In contrast in HF7c CG 1945 69 2 and AH109 the entire H S3 promoter including bothTATA boxes was replaced by the entire GAL7 promoter leading to tight regulation of the H S3 reporter in those strains Feilotter et a 1994 The HIS3 reporter plasmids pHISi and pHISi 1 used in the Matchmaker One Hybrid System also have both of the HIS3 TATA boxes present in the minimal promoter By inserting a cis acting element in the MCS the regulated TATA box TR can be affected but there is still a significant amount of constitutive leaky expression due to the H S3TC The leaky H S3 expression of these one hybrid plasmids is first used to help construct H S3 reporter
57. ase activity and the K of an interaction Estojak etal 1995 Furthermore quantitative data cannot be compared between different host strains having different lacZ reporter constructs In fact due to promoter strength differences it may be possible to quantitate the relative strength of interactions in some yeast strains e g Y190 Y187 but not in others e g CG 1945 or HF7c See Chapter 1 for a discussion of the promoters e The liquid assays described here use one of three substrates ONPG CPRG or a chemiluminescent substrate Galacton Star The three substrates differ in their relative cost sensitivity and reproducibility See Table III a Galactosidase Assays The a Gal Quantitative Assay is a sensitive colorimetric method for the detection and quantitation of yeast a galactosidase activity resulting from expression of the MEL1 reporter gene in our GAL4 based Matchmaker two hybrid systems MELT is a member of the GAL gene family which as a group facilitates the uptake and utilization of galactose by the cell Upon binding of GAL4 to the MEL1 upstream activating sequence MEL1 UAS the MEL7 gene product a galactosidase is actively Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 23 Yeast Protocols Handbook VI a and p Galactosidase Assays continued expressed and secreted to the periplasmic space and culture medium where it catalyzes the hydrolysis of melibiose to galact
58. asmid Templates 1 Optimization of thermal cycling parameters The optimal cycling parameters will vary with different templates primers experimental protocols tubes and thermal cyclers Refer to the LD Insert Screening Amplimers User Manual Ausubel et al 1995 or Roux 1995 for suggestions on optimizing PCR conditions In some cases touchdown PCR may be needed We have found that touchdown PCR significantly improves the specificity of many PCR reactions in a wide variety of applications Don et al 1991 Roux 1995 Briefly touchdown PCR involves using an annealing extension temperature thatis several degrees typically 3 10 C higher than theT of the primers during the initial PCR cycles typically 5 10 The annealing extension temperature is then reduced to the primerT for the remaining PCR cycles The change can be performed either in a single step or in increments over several cycles for example use 72 C for the first five cycles 70 C for the next 5 cycles and 68 C for the remaining cycles 2 Primer design Primer design is the single largest variable in PCR applications and the single most critical factor in determining the success or failure of PCR reactions For best results we recommend that you use LD Insert Screening Amplimers from Clontech However if you design your own primers be sure to use sequences flanking the MCS Always check and recheck your primer design before constructing or ordering primers
59. assay Breeden 8 Nasmyth 1985 used to measure B galactosidase activity is primarily used to screen large numbers of cotransformants that survive the HIS3 growth selection in a GAL4 two hybrid or one hybrid library screeening It can also be used to assay for an interaction between two known proteins in a GAL4 two hybrid system The in vivo agar plate assay is primarily used to screen large numbers of cotransformants for the expression of the lacZ reporter gene in a LexA two hybrid library screening when the reporter gene is maintained on an autonomously replicating plasmid The in vivoassay works for LexA transformants because of the lacZ reporter plasmid s high copy number and because of the preamplification step that normally precedes the 8 galactosidase assay in this system Please refer to the Matchmaker LexA Two Hybrid User Manual for more information on library screening Because of its relatively low sensitivity the in vivo agar plate assay is not suitable for screening transformants in a GAL4 based two hybrid assay or in a LexA based two hybrid assay when the reporter gene has been integrated into the host genome Liquid cultures are assayed for B galactosidase to verify and quantify two hybrid interactions Because of their quantitative nature liquid assays can be used to compare the relative strength of the protein protein interactions observed in selected transformants However there is no direct correlation between B galactosid
60. ates add agar 20 g L prior to autoclaving After autoclaving allow medium to cool to 55 C Then add the following 1 ml of 50 mg ml ampicillin stock e 1 ml of 1 0 M thiamine HCl stock e 100 ml of an appropriate sterile 10X DO stock solution In addition for HB101 cells only 4ml of a 10 mg ml stock of proline e Stock solutions for use with M9 or LB media Clontech Laboratories Inc 56 Ampicillin 50 mg ml in H O Store at 4 C no longer than 1 month Thiamine HCI 1 M filter sterilized Proline 10 mg ml filter sterilized 10X DO stock solution Appendix C A Protocol No PT3024 1 Version No PR742227 www clontech com Yeast Protocols Handbook APPENDIX D Solution Formulations A For Preparation of Protein Extracts e Protease Inhibitor Solution concentrated Always prepare solution fresh just before using Place on ice to prechill Type of protease s To prepare 688 ul inhibited Pepstatin A 0 1 mg ml 66 ul of a 1 mg ml stock solution Carboxyl proteases Sigma Cat No P4265 in DMSO Leupeptin 0 03 mM 2 ul of a 10 5 mM stock solution Some thiol and Sigma Cat No L2884 serine proteases Benzamidine 145 mM 500 ul of a 200 mM stock solution Trypsin plasmin Sigma Cat No B6506 and thrombin Aprotinin 0 37 mg ml 120 ul of a 2 1 mg ml stock solution Some serine Sigma Cat No A6279 proteases Store the individual stock solutions as directed on the labels and follow label precautions P
61. ay you can dilute the media supernatant before transferring an aliquot to the reaction tube or well Remember to correct for individual sample volumes and dilutions when tabulating final results at Step 11 5 Prepare a sufficient amount of Assay Buffer for all samples including controls and allow to equilibrate to room temperature Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook VI a and p Galactosidase Assays continued For each 1 ml assay you will need 24 ul Assay Buffer e For each 200 ul assay 96 vvell microtiter plate format you will need 48 ul Assay Buffer Note VVe recommend assaying each sample in triplicate Be sure to include positive and negative controls in your assay Also include a reagent blank in each assay to calibrate the spectrophotometer prior to reading the OD of your samples A reagent blank is composed of sterile unused culture media Assay Buffer and Stop Solution combined according to Steps 6 9 Assay Scale 200 ul 1 ml 6 Transfer cell culture medium supernatant from Step 4 16 ul 8 ul into a 1 5 ml microcentrifuge tube or into a well of a clear microtiter platea 7 Add Assay Buffer to each sample 48 ul 24 ul 8 Incubate at 30 C for 60 min Be sure to cover microtiter plates with a lid or parafilm to prevent evaporation 9 Terminate the reaction by adding Stop Solution 136 ul of 10X 960 ul of 1X Note Use 1X Stop Sol
62. aymond J amp Bloch W 1992 Prevention of pre PCR mispriming and primer dimerization improves low copy number amplifications Nucleic Acid Res 20 1717 1723 Clontech Laboratories Inc www clontech com Protocol No PT3024 1 46 Version No PR742227 Yeast Protocols Handbook X References continued Chuang S E Chen A L amp Chao C C 1995 Growth of E coliat low temperature dramatically increases the transformation frequency by electroporation Nucleic Acids Res 23 1641 R T Bechtel L J Videler J A Eron J J Gorczyca P amp Kaplan J C 1991 Maximizing sensitivity and specificity of PCR by preamplificiation heating Nucleic Acids Res 19 3749 Don R H Cox P T Wainwright B J Baker K amp Mattick J S 1991 Touchdown PCR to circumvent spurious priming during gene amplification Nucleic Acids Res 19 4008 Dower W J Miller J F amp Ragsdale W W 1988 High efficiency transformation of E coli by high voltage electroporation Nucleic Acids Res 16 6127 6145 Durfee T Becherer K Chen P L Yeh S H Yang Y Kilbburn A E Lee W amp Elledge S J 1993 The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit Genes Devel 7 555 569 Ebina Y Takahara Y Kishi F amp Nakazawa A 1983 LexA protein is a repressor of the colicin E1 gene J Biol Chem 258 13258 13261 Estojak J Brent R amp Gol
63. c Acids Res 23 4924 4925 Liu 4 VVilson T E Milbrandt J amp Johnston M 1993 Identifying DNA binding sites and analyzing DNA binding domains using a yeast selection system In Methods A Companion to Methods in Enzymology 5 125 137 Louret O F Doignon F amp Crouzet M 1997 Stable DNA binding yeast vector allowing high bait expression for use in the two hybrid system BioTechniques 23 816 819 Luo Y Vijaychander S Stile J amp Zhu L 1996 Cloning and analysis of DNA binding proteins by yeast one hybrid and one two hybrid systems Bio Techniques 20 564 568 Mahadevan S 8 Struhl K 1990 Tc an unusual promoter element required for constitutive transcription of the yeast HIS3 gene Mol Cell Biol 10 4447 55 Marcil R 8 Higgins D R 1992 Direct transfer of plasmid DNA from yeast to E coli by electroporation Nucleic Acids Res 20 917 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor Laboratory Cold Spring Harbor NY Miller J H 1992 In A Short Course in Bacterial Genetics Cold Spring Harbor Laboratory Press Cold Spring Harbor p 74 Post Beittenmiller M A Hamilton R W amp Hopper J E 1984 Regulation of basal and induced levels of the WEL 7 transcript in Saccharomyces cerevisiae Mol Cell Biol 4 1238 1245 Pringle J R Roach J R amp Jones E W eds 1997 The Molecular and Cellular Biology of the Yeast Saccharomyces Cell Cycle and Cell Bi
64. carries the euB mutation Bolivar amp Backman 1979 may be used to select for yeast plasmids bearing the LEU2 marker only For nutritional selection of KC8 and HB101 transformants on M9 minimal medium add a 1X mixture of amino acids i e dropout DO supplement lacking the specific nutrient that will allow selection of the desired plasmid Appendix E The same DO supplements used for yeast SD medium can be used to supplement M9 minimal medium see Appendix C for recipes Because ofits auxotrophic mutations KC8 requires His Leu Trp and thiamine for growth on minimal medium unless one of these nutrients is specifically omitted for the selection e HB101 requires leucine proline and thiamine for growth on minimal medium unless one of these nutrients is specifically omitted for the selection note that HB101 is streptomycin resistant Although optional we recommend including ampicillin 50 ug ml inthe medium to reduce background growth Any of the common E colihost strains e g DH5a JM109 may be used if you prefer to select transformants by resistance to ampicillin rather than using a nutrional selection However because both the DNA BD and AD plasmids will be represented in the E coli transformant population and not necessarily in equal proportions many transformant colonies will need to be screened for the presence of the desired plasmid s The plasmids can be distinguished by restriction enzyme digestion or
65. centrifuging at 14 000 rpm for 5 min iv Carefully pour off the supernatant and resuspend pellets in the residual liquid total volume 50 ul c For semi automated handling of a large number of samples i Place a large 2 4 mm fresh 2 4 day old yeast colony into 0 5 ml ofthe appropriate SD liquid medium in separate wells of a 96 tube microtiter array Vortex each tube vigorously to resuspend the cells Alternatively use 0 5 ml of an overnight SD liquid culture instead of a yeast colony ii Using acentrifuge adapted for multiwell plates centrifuge the entire array at 1 000 x g for 5 min to pellet the cells iii Carefully pour or draw off supernatants and resuspend pellets in the residual medium 50 ul by vortexing or pipetting up and down 2 Add 10 uloflyticase solution to each tube Thoroughly resuspend the cells by vortexing or repeatedly pipetting up and down 3 Incubate tubes at 37 C for 30 60 min with shaking at 200 250 rpm Optional Check a drop of the cell suspension under a phase contrast microscope 400X for the progress of cell lysis by adding a drop of 20 SDS to the side of the coverslip As they come into contact with the SDS most cells should lose their refractile appearance and appear as ghost like spheroplasts If there are still many intact cells present incubate the samples for another 30 min 4 Add 10 ul of 20 SDS to each tube and vortex vigorously for 1 min to mix 5 Put the samples through one
66. d Select for transformants on the appropriate SD dropout medium For each plasmid of interest to be tested set up pairwise yeast matings with transformants containing control plasmids Use either the standard procedure Section C 2 or the procedure adapted for microtiter 96 well plates Section C 3 2 Yeast mating procedure standard a b Pick one colony of each type to use in the mating Use only large 2 3 mm fresh lt 2 months old colonies from the working stock plates Place both colonies in one 1 5 ml microcentrifuge tube containing 0 5 ml of YPD medium Vortex tubes to completely resuspend the cells Incubate at 30 C overnight 20 24 hr with shaking at 200 rpm Spread 100 ul aliquots of the mating culture on the appropriate SD minimal media Use double dropoutto select for both plasmids and triple dropout to select for diploids in which a positive two hybrid interaction is occurring Proceed to step 4 below 3 Yeast mating procedure microtiter plate version If you have many plasmids of interest to mate to several control strains it may be more efficient to set up the matings in separate wells of a sterile flat bottom microtiter plate In between steps keep plate covered with a sterile lid a b g Aliquot 160 ul of YPD medium to each well For each plasmid of interest to be tested place a single transformant colony in a 1 5 ml microcentrifuge tube containing 1 ml of YPD Vigorously vortex the tube t
67. d System utilize the full length GAL7 promoter to drive fusion protein expression Because the LexA system host strain is wild type for GAL4 and GAL80 fusion protein expression is regulated by glucose and galactose Clontech Laboratories Inc www clontech com Protocol No PT3024 1 8 Version No PR742227 Yeast Protocols Handbook ll Introduction to Yeast Promoters continued TABLE II YEAST PROMOTER CONSTRUCTS IN THE V ATCHMAKER CLONING VECTORS Regulation Signal Relative Protein Strength on Vectors Promoter Expression Level Western blot p LexA pGAD GH 1 full length Ethanol repressed High pAS2 1 pAS2 pGADT7 pACT2 pACT ADH1 410 bp Constitutive medium pGAD GL ADH1 410 bp Constitutive low weak pGAD424 pGAD10 Constitutive very low not detectable pGBT9 pGBKT7 ADH1 700 bp Consitutive high pB42AD pGilda GAL1 full length Repressed by glucose not detectable 4 induced high level by galactose 4 4 p8op lacZ GAL1 minimal Not regulated by glucose no data or galactose 3 See Appendix E for vector references gt Unpublished data obtained at Clontech Laboratories using the appropriate GAL4 domain specific mAb Cat No 630402 or Cat No 630403 Soluble protein extracts were prepared from CG 1945 transformed with the indicated plasmid Samples equivalent to 1 OD unit of cells were electrophoresed and then blotted to nitrocellulose filters The blots were probed with either
68. dase Filter Assays Z buffer Na HPO 7 16 1 g L NaH PO H O 5 50 g L KCI 0 75 g L MgSO 7H O 0 246 g L Adjust to pH 7 0 and autoclave Can be stored at room temperature for up to 1 year X gal stock solution Dissolve 5 bromo 4 chloro 3 indolyl 8B D galactopyranoside X GAL Cat No 8060 1 in N N dimethylformamide DMF at a concentration of 20 mg ml Store in the dark at 20 C Z buffer X gal solution 100 ml Z buffer 0 27 ml mercaptoethanol B ME Sigma Cat No M 6250 1 67 ml X gal stock solution D For Liquid b galactosidase Assays with ONPG as Substrate e Z buffer see preceding section for recipe Z buffer with B mercaptoethanol To 100 ml of Z buffer add 0 27 ml of B mercaptoethanol ONPG t o nitrophenyl B D galactopyranoside Sigma Cat No N 1127 4 mg ml in Z buffer Adjust to pH 7 0 and mix well Notes e ONPG requires 1 2 hr to dissolve e Prepare solution fresh before each use Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 59 Yeast Protocols Handbook APPENDIX D Solution Formulations continued E For Liquid b galactosidase Assays with CPRG as Substrate Buffer 1 To prepare 100 ml of solution HEPES 2 38 g NaCl 0 9g L Aspartate hemi Mg salt Sigma Cat No A 9506 0 065 g BSA 1 0 g Tween 20 50 0 ul Dissolve the above components in 75 ml of deionized H O Adjust pH to 7 25 7 30 then bring volume to 100 ml Filter sterilize
69. dase positive colonies color development occurs within seconds weak to moderate reactions take several hours to develop 16 Centrifuge samples at 14 000 rpm for 1 min to pellet cell debris 17 Transfer samples to fresh tubes 18 Zero the spectrophotometer using the buffer blank and measure the OD of the samples An OD between 0 25 and 1 8 is within the linear range of the assay 19 Calculate B galactosidase units 1 unit of B galactosidase is defined as the amount which hydrolyzes 1 umol of CPRG to chlorophenol red and D galactose per min per cell Miller 1972 Miller 1992 B galactosidase units 1000 x OD t xV x OD where t elapsed time in min of incubation V x concentration factor Asoo Of 1 ml of culture The concentration factor from Step E 7 is 5 However it may be necessary to try several dilutions of cells at this step hence different concentration factors to remain within the linear range of the assay F Liquid Culture Assay Using a Chemiluminescent Substrate Reagents and Materials Required Appropriate liquid medium Appendix C A 50 ml culture tubes Z buffer Appendix D Galacton Star reaction mixture Provided with the Luminescent B galactosidase Detection Kit Liquid nitrogen Luminometer or scintillation counter with single photon counting program Optional 96 well opaque white flat bottom microtiter plates Xenopore Cat No WBP005 Protocol No PT3024
70. detection can be performed 1 2 hr after the incubation 21 Centrifuge tubes at 14 000 rpm 16 000 x g for 1 min at 4 C If you are using microtiter plates centrifuge plates at 1 000 x g for 5 min in a specially adapted rotor Proceed directly to the appropriate detection steps for your assay Step 22 23 24 or 27 22 Detection using a tube luminometer a Turn on the tube luminometer Set the integration time for 5 sec b Calibrate the luminometer according to the manufacturer s instructions c Ifthe sample is not already in a tube suitable for luminometer readings transfer the entire solution from Step 21 to an appropriate tube Do not disturb the pellet d Place one sample ata time inthe luminometer compartmentand record the lightemission RLU as a 5 sec integral Use your blank sample as a reference when interpreting the data 23 Detection using a plate luminometer After Step 21 simply record light signals as 5 sec integrals 24 Detection using a scintillation counter a Transfer the entire solution from Step 21 to a 0 5 ml microcentrifuge tube Note Plan to use scintillation counter adaptors that keep the tubes upright b Place the tube in the washer of the scintillation counter adaptor and place the adaptor in the machine s counting rack Set the integration time for at least 15 sec Note Integration times lt 15 sec may not produce accurate results c Todetect chemiluminescent signals use a single photon count p
71. dium with no antibiotic h Incubate at 37 C for 1 hr with vigorous shaking 250 rpm i Pellet cells by centrifuging at 2 500 rpm for 5 min in a table top centrifuge j Discard supernatant and resuspend pellet in residual liquid k Plate cells on appropriate medium LB amp or supplemented M9 Incubate plates at37 C for 24 hr LB amp selection only or for 36 48 hr for nutritional selection on M9 medium Typically 10 100 colonies will be seen on the plate for a successful transformation using plasmid isolated from yeast If you do not recover any colonies see the Troubleshooting tips below m If you performed a parallel transformation using the control pUC19 DNA calculate the transformation efficiency The competent cells should have been transformed with an efficiency of gt 1 x 10 cfu ug See Section V E 23 for a sample calculation n See Section C 3 for tips on plasmid isolation 3 Tips on Isolating plasmid DNA from the E coli transformants a Use a standard plasmid mini prep procedure to isolate plasmid DNA from the E coli transformants Sambrook et al 1989 Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 37 Yeast Protocols Handbook VILVVorking With Yeast Plasmids continued Notes e f you are using an endA bacterial strain such as KC8 or HB101 as the host strain extra care must be taken when preparing plasmid DNA because of the presence of endonuclease
72. e See your system specific User Manual or Appendix E for media recommendations Be sure to plan for enough plates for the control transformations and platings Allow SD agar plates to dry unsleeved at room temperature for 2 3 days or at 30 C for 3 hr prior to plating any transformation mixtures Excess moisture on the agar surface can lead to inaccurate results due to uneven spreading of cells or localized variations in additive concentrations e Appropriate plasmid DNA in solution check amounts required e Appropriate yeast reporter strain for making competent cells check volume of competent cells required Steps 1 11 of Section V E will give you 1 5 ml enough for 14 15 small scale transformations Herring testes carrier DNA Appendix D B e Sterile PEG LiAc solution Prepare only the volume needed immediately prior to use from 10X stocks Appendix D B e 100 DMSO Dimethyl sulfoxide Sigma Cat No D 8779 Sterile 1XTE buffer Prepare from 10XTE buffer Appendix D B Sterile glass rod bent Pasteur pipette or 5 mm glass beads for spreading cells on plates Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 19 Yeast Protocols Handbook V Yeast Transformation Procedures continued C Tips for a Successful Transformation Fresh one to three week old colonies will give best results for liquid culture inoculation A single colony may be used for the inoculum if it is 2 3 mm
73. e the 200 and 1 ml reactions the final pHs and therefore the molar absorbtivities are different for the two formats Determined at Clontech using a SPECTRAmax Microplate Spectrophotometer and Corning Costar UV transparent flat bottom plates Corning Cat No 3635 Some microplate readers have pathlength correction capabilities to normalize absorbance values to those obtained when the light pathlength is 1 cm e g using 1 5 ml cuvettes With pathlength correction on the molar absorbtivity s of p nitrophenol at 410 nm in the 200 ul format was determined to be 20 3 ml umol The well diameter and therefore the light path b in other brands of 96 well plates may differ from that of the Corning plates used for these determinations To determine s x b in other plates construct a plot of A versus concentration of p nitrophenol PNP as follows Using a 10 umol ml standard solution of p nitrophenol Sigma Cat No 104 1 make 1 2 serial dilutions in water down to 0 02 umol ml PNP Use these serial dilutions in place of medium supernatant in Step 6 above Then follow Steps 7 9 and 10 as directed omit Step 8 At Step 7 use Assay Buffer that has been prepared by combining 2 volumes of 1X NaOAc with 1 volume of H O not PNP a Gal Finally plot Az versus concentration of PNP According to the Beer Lambert Law the proportionality constant e x b is equal to the slope of the straight line defined by these data 4 Determined at Clontech usin
74. emis E A 1995 Correlation of two hybrid affinity data with in vitro measurements Mol Cell Biol 15 5820 5829 Feilotter H E Hannon G J Ruddel C J amp Beach D 1994 Construction of an improved host strain for two hybrid screening Nucleic Acids Res 22 1502 1503 Finley Jr R L amp Brent R 1994 Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators Proc Natl Acad Sci USA 91 12980 12984 Flick J S amp Johnston M 1990 Two systems of glucose repression of the GAL7 promoter in Saccharomyces cerevisiae Mol Cell Biol 10 9 4757 4769 Gietz D St Jean A Woods R A 8 Schiestl R H 1992 Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res 20 1425 Gimeno R E Espenshade P amp Kaiser C A 1996 COPII coat subunit interactions Sec24p and Sec23p bind to adjacent regions of Sec16p Mol Biol Cell 7 1815 23 Giniger E Varnum S M amp Ptashne M 1985 Specific DNA binding GAL4 a positive regulatory protein of yeast Cell 40 767 74 Giniger E amp Ptashne M 1988 Cooperative DNA binding of the yeast transcriptional activator GAL4 Proc Natl Acad Sci USA 85 382 386 Golemis E A Gyuris J amp Brent R 1996 Analysis of protein interactions and interaction trap two hybrid systems to identify interacting proteins In Current Protocols in Molecular Biology John Wiley amp Son
75. ensitive than ONPG e Potential drawbacks Less reproducible than ONPG for strong positive colonies because of CPRGss fast reaction rate Liquid culture Chemiluminescent VI F e For assaying asmallnumber of selected transformants Galacton Star eThe most sensitive B gal substrate e Potential drawbacks Relatively expensive Requires luminometer or scintillation counter Can give high background Summary Relative sensitivity of the five types of b galactosidase assays Least sensitive Most sensitive X gal ONPG CPRG X gal Galacton Star in agar plates liquid assay liquid assay filter assay liquid assay Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 25 Yeast Protocols Handbook VI a and p Galactosidase Assays continued B In vivo Plate Assay Using X gal in the Medium For LexA Systems only Reagents and Materials Required e Appropriate SD agar plates containing X gal 80 mg L and 1X BU salts Appendix C A Notes BU salts are included in the medium to maintain the optimum pH for B galactosidase and to provide the phosphate needed for the assay The X gal should be incorporated into the medium before the plates are poured If the X gal is spread over the surface of the agar plates it can result in uneven distribution and thus localized variations in X gal concentration Also the extra liquid on the plate surface from spreading the X gal may lead to
76. er strains cotransformed with the positive controls for a two hybrid interaction give a positive blue signal within 60 min CG 1945 cotransformed with the control plasmids may take an additional 30 min to develop If the controls do not behave as expected check the reagents and repeat the assay 10 Identify the B galactosidase producing colonies by aligning the filter to the agar plate using the orienting marks Pick the corresponding positive colonies from the original plates to fresh medium If the entire colony was lifted onto the filter incubate the original plate for 1 2 days to regrow the colony D Liquid Culture Assay Using ONPG as Substrate Reagents and Materials Required Appropriate liquid medium Appendix C A 50 ml culture tubes Z buffer Appendix D Z buffer B mercaptoethanol Appendix ONPG Appendix D 1 M Na CO Liquid nitrogen 1 Prepare 5 ml overnight cultures in liquid SD selection medium as described in Chapter 11 Use the SD medium appropriate for your system and plasmids Note Be sure to use SD medium that will maintain selection on the plasmids used 2 On the day of the experiment dissolve ONPG at 4 mg ml in Z buffer Appendix D with shaking for 1 2 hr 3 Vortex the overnight culture tube for 0 5 1 min to disperse cell clumps Immediately transfer 2 ml of the overnight culture to 8 ml of YPD except for the LexA System Note For the LexA System use the appropriate SD Gal Raff induc
77. erest has integrated into the yeast chromosome and therefore cannot replicate autonomously when introduced into E coli If the plasmid s insert can be amplified by PCR Chapter VIII it may be possible to recover the insert by subcloning from the PCR product e The plasmid may encode a protein that is toxic to E coli Again it may be possible to recover the insert by subcloning the PCR amplified fragment Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook VIII Analysis of Yeast Plasmid Inserts by PCR A General Information Sometimes a two hybrid library screening results in many even hundreds of positive candidate clones However a few abundant insert sequences may account for the majority Sorting colonies into groups will eliminate duplicates bearing the same plasmid insert and will save time in the subsequent analysis The cDNA inserts from all plasmids encoding candidate interacting proteins can be amplified by PCR and sorted into groups based on restriction digestion patterns After colonies have been sorted a representative clone from each group can be transferred to a new master plate for further analysis To ensure efficient amplification of all inserts regardless of size we strongly recommend the use of long distance LD PCR Barnes 1994 Cheng et al 1994 with the Advantage 2 Polymerase Mix Cat No 639201 The Advantage 2 PCR Kit Cat No 639206
78. ese same considerations are important for the subsequent transfer of proteins to the nitrocellulose membrane where transfer buffer composition temperature duration of transfer and the assembly of the blotting apparatus can all have profound effects on the quality of the resultant protein blot The following troubleshooting tips pertain to the isolation of protein from yeast Information on running polyacrylamide protein gels and performing Western blots is available in published laboratory manuals e g Sambrook et al 1989 or Ausubel et al 1987 96 1 Few or no immunostained protein bands on the blot e The transfer of protein bands to the blot may be confirmed by staining the blot with Ponceau S e The presence of protein bands in the gel before transfer may be confirmed by staining a parallel lane of the gel with Coomassie blue Note that once a gel has been stained with Coomassie blue the protein bands will not transfer to a blot The extent of cell wall disruption can be determined by examining a sample of treated cells under the microscope Incomplete cell lysis will lower the protein yield 2 Several bands appear on the blot where a single protein species is expected e Protein degradation and or proteolysis may have occurred during sample preparation Additional protease inhibitors may be used as desired Also make sure that in Steps C 8 a and D 12 boiling the protein extracts the samples are placed into a water bath
79. fore recommend that you obtain several different exposures Qualitative Liquid Assay Using Galacton Star as the Substrate This alternative cell preparation method directly detects B galactosidase activity in resuspended yeast colonies It is recommended for detecting extremely weak 2 transcriptional signals that cannot be detected by X gal filter assays For a result itis more labor intensive than a filter assay However because of its greater sensitivity it is less likely to give a false negative result 1 Grow colonies on the appropriate SD selection medium 2 Transfer an entire large 2 3 mm fresh 2 4 day old colony to a 0 5 ml tube containing 50 ul of Z buffer If colonies are small use several At the same time prepare a master Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 31 Yeast Protocols Handbook Vi a and Galactosidase Assays continued or reference plate of the colonies to be assayed 3 Completely resuspend the colony in the Z buffer by repeatedly pipetting up and down 4 Place tubes in liquid nitrogen for 0 5 1 min to freeze the cells 5 Continue with Step 13 of the main procedure VI F above 6 Compare results with those of the negative control G a Gal Quantitative Assay Reagents and Materials Required Appropriate liquid synthetic dropout SD culture medium Appendix C A 50 ml culture tubes PNP a Gal Solution 100 mM 10X Stop Solution Appe
80. g 1 5 ml cuvettes Protocol PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 Yeast Protocols Handbook VIl Working With Yeast Plasmids A General Information Isolating plasmid DNA from yeast is not trivial primarily because of the tough cell wall Furthermore the relatively large size gt 6 kb and low copy number 50 cell of some yeast plasmids results in very low DNA yields regardless of the plasmid isolation method used In addition plasmid DNA isolated from yeast is often contaminated by genomic DNA because yeast contain 3X as much genomic DNA as E coli and the isolation method breaks the yeast chromosomes and releases them from cellular material There are several yeast plasmid isolation procedures currently in use The various protocols differ primarily in the method used to break the cell walls Here we provide the protocol that we optimized for ourYeastmakerYeast Plasmid Isolation Kit Cat No 630441 This procedure which was modified from the method of Ling et al 1995 uses extensive digestion with lyticase to weaken the cell walls and SDS to burst the resulting spheroplasts The DNA preps can be cleaned up using either CHROMA SPIN Columns or phenol chloroform extraction followed by ethanol precipitation If CHROMA SPIN Columns are used this method takes lt 2 hr from cell pellets to purified plasmid and is simple enough to be easily adapted for processing many samp
81. g so use electrotransformation rather than chemical transformation higher transformation efficiencies are usually obtained with electroporation e Use competent cells that are known to be transformed with a very high efficiency Both chemically competent and electrocompetent cells are available from Clontech b Ifyou try the measures recommended in Section 4 a above and still do not recover any E coli transformants the problem may be the plasmid preparation or the plasmid itself e The yeast plasmid preparation may have no plasmid DNA in it Check the medium you used for the overnight cultures It is important to use a medium that maintains selection on the desired plasmid The working stock plate used as your inoculum source should also keep selection on the plasmid When you repeat the plasmid isolation procedure be sure to include the freeze thaw cycle at Step VII B 5 to ensure complete cell lysis e Check the concentration of total DNA in your plasmid prep using absorbance at 260 nm or by running a small sample 10 ul on a gel Although plasmid DNA makes up only a small fraction of the total DNA you can at least confirm that you have DNA in your prep The larger chromosomal DNA fragments should be visible on a 1 agarose EtBr gel The limit of detection with EtBr staining is 4 ng Sambrook et al 1989 Appendix E 5 e Even if you have a substantial amount of DNA in your prep there is a remote possibility that the plasmid of int
82. haromyces cerevisiae Biochem Biophys Res Commun 27 299 304 Stargell L A amp Struhl K 1996 Mechanisms of transcriptional activation in vivo two steps forward Trends Genet 12 8 311 315 Tirode F Malaguti C Romero F Attar R Camonis J amp Egly J M 1997 A conditionally expressed third partner stabilizes or prevents the formation of a transcriptional activator in a three hybrid system J Biol Chem 272 22995 22999 Tornow J amp Santangelo G G 1990 Efficient expression of Saccharomyces cerevisiae glycolytic gene ADH7 is dependent upon a cis acting regulatory element UAS pgo found initially in genes encoding ribosomal proteins Gene 90 79 85 van Aelst L Barr M Marcus S Polverino A amp Wigler M 1993 Complex formation between RAS and RAF and other protein kinases Proc Natl Acad Sci USA 90 6213 6217 Weisman L S Bacallao R amp Wickner W 1987 Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle J Cell Biol 105 1539 1547 West R W Jr Yoccum R R amp Ptashne M 1984 Saccharomyces cerevisiae GAL1 GAL10 Divergent Promoter Region Location and Function of the Upstream Activating Sequence UAS Molecular and Cellular Biology 4 2467 2478 Yoccum R R 1987 The GAL1 7 and 10 upstream activator sequences are not enhancers in Biological Research on Industrial Yeasts Vol 3 Stewart G Russell
83. he mixed plasmid DNA from yeast use it to transform E coli isolate the desired plasmid from E colitransformants and transform the desired yeast host strain with the isolated plasmid DNA Alternatively the yeast cotransformantstrain can be grown for several generations on SD medium that maintains selection onthe desired plasmid only as described in Section B 1 below The search for yeast segregants can be significantly accelerated if you are working with a cycloheximide resistant yeast host strain and the unwanted plasmid confers sensitivity to cycloheximide as described in Section B 2 below Cycloheximide counterselection is an option with the Matchmaker Two Hybrid System 2 Cat No K1604 1 but cannot be used with the host strains provided with Pretransformed Matchmaker Libraries or the original Matchmaker System Cat No K1605 1 1 Segregation by natural loss of an unselected plasmid a Cultureindividual cotransformantcolonies separately in 3 mloftheappropriate SD liquid selection medium for 1 2 days at 30 C with shaking 230 250 rpm The medium must maintain selection on the plasmid of interest but not on the plasmid you wish to lose Under these conditions the plasmids that are not selected for are lost at a rate of 10 20 per generation Refer to Appendix E for information on yeast plasmid transformation selection markers b Spread a diluted sample of this liquid culture on agar plates that will select for the desired plasmid
84. hether you are selecting for colonies in which two hybrid proteins are interacting whether and to what extent the host strain is leaky for reporter gene expression whether you want to induce protein expression from the regulated GAL7 promoter whether you intend to perform in vivo agar plate B galactosidase assays for lacZ reporter expression in the LexA Two Hybrid System Please refer to your system specific User Manual for further information on choosing the appropriate SD selection media for particular plasmids host strains and applications Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 11 Yeast Protocols Handbook IV Preparation of Yeast Protein Extracts A General Information We provide two alternative protocols for the preparation of protein extracts from yeast The results i e protein yield and quality will vary depending on the protein and may be more successful with one protocol than with the other Because it is difficult to predict which procedure will give better results we provide two protocols for comparison The cell culture preparation method Section B is the same for both protein extraction procedures Both extraction procedures address the two most challenging aspects of isolating proteins from yeast 1 disrupting yeast cell walls and 2 inhibiting the many endogenous yeast proteases Yeast cell walls are tough and must be disrupted by a combination of physica
85. hmaker Two Hybrid and One Hybrid Systems and Matchmaker Libraries The Yeast Protocols Handbook is especially useful for researchers who wish to use yeast as a vehicle for their molecular biology experiments but have little or no prior experience working with yeast For novice and experienced users alike the Yeast Protocols Handbook will help you obtain the best possible results with your Matchmaker and other yeast related products from Clontech This Handbook includes e detailed information on culturing and handling yeast information on the yeast promoters used in the Matchmaker Systems e two protocols for preparing protein extracts from yeast quantitative and qualitative B galactosidase assays for use with lacZ yeast reporter strains e a simple optimized protocol for isolating plasmids from yeast e PCR amplification and yeast colony hybridization protocols for the rapid analysis of positive clones obtained in a library screening e a small scale lithium acetate yeast transformation protocol e additional protocols for working with certain yeast plasmids and host strains The special application of yeast transformation for one and two hybrid library screening is covered in detail in each product specific User Manual The special application of yeast mating for library screening is covered in the Pretransformed Matchmaker Libraries User Manual About our yeast based products The Matchmaker GAL4 Two Hybrid Systems Cat No K1
86. ich RNA polymerase complex binds and initiates transcription of an adjacent structural gene or gene cluster In yeast the promoter is typically comprised of at least one TATA box and other closely associated cis regulatory elements e g UASs prototroph A strain of yeast or other microorganisms that will proliferate even if a particular nutrient is not supplied in the medium For example Trp yeast strains are protototrophic for Trp they can synthesize their ownTrp from other biomolecules and do not require it in the medium A prototrophictransformation marker or reporter gene can be used to complement the corresponding auxotrophic allele in another strain segregation Genetically the production from a single cell of two daughter cells having distinct genotypes and phenotypes due to the separation of two alleles of a gene In yeast this can occur during sporulation or in transformant clones as a result of loss of a plasmid trans acting element A gene that controls transcriptional activity of another gene through a diffusable gene product protein such as a repressor or activator transformation The process of introducing foreign DNA into a cell transformation markers Genetic alleles whose phenotypes identify the presence of a plasmid introduced into acell typically such markers are genes that complement a nutritional requirement or confer resistance to an antibiotic UAS Upstream Activating Sequence yeast DNA sequences that con
87. idization B Generating Yeast Plasmid Segregants C Yeast Mating References Matchmaker and Related Products APPENDICES A B C m Glossary ofTechnical Terms Yeast Genetic Markers Used in the Matchmaker Systems Media Recipes A Yeast Media B E coli Media Solution Formulations Plasmid Information Yeast Host Strain Information Clontech Laboratories Inc www clontech com 2 10 12 12 12 13 15 17 18 18 19 20 20 20 22 23 23 25 25 26 27 28 32 34 34 34 36 39 39 39 42 42 43 44 46 49 50 52 53 53 56 57 61 64 Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook Table of Contents continued List of Tables Table 1 Yeast Promoter Constructs Used to Regulate Reporter Gene Expression in Matchmaker Plasmids and Host Strains 6 Table Il Yeast Promoter Constructs in the Matchmaker Cloning Vectors 9 Table Ill Comparison of 8 galactosidase Assays 25 Table IV Selected Yeast Genes and Their Associated Phenotypes 52 Table V Matchmaker Reporter Genes and Their Phenotypes 52 Table VI Matchmaker Two Hybrid System Cloning Vectors 61 Table VII Matchmaker Two Hybrid System Reporter and Control Plasmids 62 Table VIII Matchmaker One Hybrid System Cloning Reporter amp Control Plasmids 63 Table IX Yeast Reporter Strains in the Matchmaker One and Two Hybrid Systems 64 List of Figures Figure 1 Sequence of GAL4 DNA BD recognition sites in the GAL1 G
88. iency of the single plasmids from the number of transformants growing onthe appropriate control plates If the two types of plasmids separately gave transformation efficiencies gt 10 cfu ug switch to sequential transformation If the transformation efficiency for one or both of the separate plasmids is lt 10 cfu ug several causes are possible 1 Suboptimal plasmid preparation Repeat the transformation using more up to 0 5 ug of the plasmid DNA that had the low transformation efficiency Check the purity of the DNA and if necessary repurify it by ethanol precipitation before using it again Suboptimal carrier DNA If you are not already doing so useYeastmaker Carrier DNA which is available separately Cat No 630440 or as part of theYeastmakerYeastTransformation System Cat No 630439 and has been optimized for high transformation efficiencies in this system If transformation efficiencies are declining in successive experiments the carrier DNA may be renaturing Reboil the carrier DNA for 20 min and then chill it quickly in an ice water bath Suboptimal yeast competent cells Make sure that the expansion culture Step E 6 was in log phase growth at the time the cells were harvested for making competent cells If the overnight culture Step E 4 or expansion culture Step E 6 grew slower than expected or not at all start over at Step E 1 by preparing a fresh overnight culture Failure to thoroughly disperse the
89. in diameter Scrape the entire colony into the medium If colonies on the stock plate are smaller than 2 mm scrape several colonies into the medium See Chapter III A for further information on starting liquid cultures from colonies and from a liquid culture inoculum Vigorously vortex liquid cultures to disperse the clumps before using them in the next step The health and growth phase of the cells at the time they are harvested for making competent cells is critical for the success of the transformation The expansion culture Step E 6 should be in log phase growth i e OD between 0 4 and 0 6 at the time the cells are harvested If they are not see the Troubleshooting guide Section V F When collecting cells by centrifugation a swinging bucket rotor results in better recovery of the cell pellet For the highest transformation efficiency as is necessary for library screening use competent cells within 1 hr of their preparation If necessary competent cells can be stored after Step E 11 at room temperature for several hours with a minor reduction in competency To obtain an even growth of colonies on the plates continue to spread the transformation mixtures over the agar surface until all liquid has been absorbed Alternatively use 5 mm sterile glass beads 5 7 beads per 100 mm plate to promote even spreading of the cells D Integrating Plasmids into the Yeast Genome Im portant Please read Section V A for guidelines
90. in yeast is typically approximately 25 bp upstream of the transcription start site Yeast TATA boxes are functionally similar to prokaryotic Pribnow boxes but are not as tightly conserved Furthermore some yeast transcription units are preceded by more than one TATA box The yeast H S3 gene for example is preceded by two different TATA boxes TR which is regulated and TC which is constitutive Mahadevan amp Struhl 1990 Yeast TATA boxes can be moved to a new location adjacent to other cis regulatory elements and still retain their transcriptional function One type of cis acting transcription element in yeast is upstream activating sequences UAS which are recognized by specifictranscriptional activators and enhance transcription from adjacent downstream TATA regions The enhancing function of yeast UASs is generally independent of orientation however it is sensitive to distance effects if moved more than a few hundred base pairs from the TATA region There may be multiple copies of a UAS upstream of a yeast coding region In addition UASs can be eliminated or switched to change the regulation of target genes UAS andTATA regions can be switched to create novel promoters The mix and match nature of yeastTATA boxes and UASs has been used to great advantage in yeast two hybrid systems to create novel promoters for the reporter genes For general references on yeast two hybrid systems see Chapter X In most cases the lacZ HIS3
91. iwith plasmids isolated from yeast because of the relatively high transformation efficiency that can be obtained This is important because of the yeast genomic DNA that is present in yeast isolated plasmids the presence of genomic DNA reduces the transformation efficiency of the plasmids However if you choose to use chemically competent cells Section C 2 it is essential that the cells be able to yield a transformation efficiency of at least 10 cfu ug of pUC19 DNA Nutritional selection of E coli transformants Inthe Matchmaker two hybrid systems cloning vectors carrying HIS3 LEU2 or TRP1 markers can be selectively rescued by complementation of the E coli hisB leuB or mutations respectively The yeast H S3 LEU2 and TRP1 genes are expressed well enough in E coli to allow this complementation Furthermore due to incompatibility of the E coli plasmid replication origins used on the different vectors only one plasmid construct will propagate in a given E coli transformant plated on selection medium Thus there is no need to screen every E coli transformant for the presence of the other unwanted plasmids If you plan to perform a nutritional selection for plasmid rescue we recommend using E coli srain KC8 which carries the hisB leuB and troC mutations K Struhl personal communication KC8 Chemically Competent Cat No 630434 and Electrocompetent Cat No 630435 Cells are available from Clontech HB101 which
92. ix E for plasmid information To prepare new glycerol stock cultures of yeast a b C Use a sterile inoculation loop to scrape an isolated colony from the agar plate Resuspend the cells in 200 500 ul of YPD medium or the appropriate SD medium in a 1 5 ml microcentrifuge tube Vortex tube vigorously to thoroughly disperse the cells Add sterile 50 glycerol to a final concentration of 25 Tightly close the cap Shake the vial before freezing at 70 C 2 To recover frozen strains and prepare working stock plates a b Streak a small portion of the frozen glycerol stock onto a YPD or appropriate SD agar plate Incubate the plate at 30 C until yeast colonies reach 2 mm in diameter this takes 3 5 days Use these colonies as your working stock Seal plates with Parafilm and store at 4 C for up to two months Streak a fresh working stock plate from the frozen stock at 1 2 month intervals If you cannot recover the strain the cells may have settled in this case thaw the culture on ice vortex vigorously and restreak The glycerol stock tube may be refrozen a few times without damaging the cells 3 To prepare liquid overnight cultures a Use only fresh lt 2 months old colonies from the working stock plate Use one large 2 3 mm diameter colony per 5 ml of medium If colonies are small or if you are inoculating a larger volume use several colonies Important Vigorously vortex the medium for 1 min
93. l and chemical means methods that utilize glycolytic enzymes are not recommended for this application because they are often contaminated with proteases Endogenous proteases must be counteracted with a cocktail of strong protease inhibitors recipe in Appendix D A If you know your protein of interest is susceptible to a protease not inhibited by the recommended cocktail add the appropriate inhibitor before using the mixture You may also wish to add other inhibitors such as sodium fluoride to prevent dephosphorylation if that is appropriate for your protein Preparation of Yeast Cultures for Protein Extraction Reagents and Materials Required YPD and appropriate SD liquid medium Recipes in Appendix C A 20 and 50 ml culture tubes Ice cold H O Dry ice or liquid nitrogen For each transformed yeast strain you wish to assay in a Western blot prepare a 5 ml overnight culture in SD selection medium as described in Section III A except use a single isolated colony 1 2 mm in diameter no older than 4 days Use the SD medium appropriate for your system and plasmids Appendix E Also prepare a 10 ml culture of an untransformed yeast colony in YPD or if possible appropriate SD medium as a negative control Vortex the overnight cultures for 0 5 1 min to disperse cell clumps For each clone to be assayed and the negative control separately inoculate 50 ml aliquots of YPD medium with the entire overnight culture
94. les simultaneously This purification method yields DNA of sufficient purity for use as a PCR template Chapter VIII or for transforming E coli Chapter VII C However if you need a large quantity of plasmid or very pure plasmid DNA such as for sequencing or restriction enzyme digestion you will have to transform E coli and prepare plasmid using standard methods Sambrook et al 1989 Plasmid rescue via complementation of E coli mutations Plasmid isolation from yeast cotransformants is complicated by the presence of two or more types of plasmids in a single yeast colony Nutritional selection of E colitransformants bearing the yeast plasmid of interest can be an efficient way to rescue one type of plasmid from a mixture of plasmids bearing different nutritional transformation markers For more information on plasmid rescue via transformation of E coli see Section VII C Plasmid Isolation from Yeast Reagents and Materials Required The Yeastmaker Yeast Plasmid Isolation Kit Cat No 630441 provides the SDS and lyticase solutions CHROMA SPIN 1000 Columns and 2 ml centrifuge tubes for use with the columns e Appropriate SD liquid or agar medium to keep selection on the plasmids Appendix C A Appendix E Sterile 1 5 ml microcentrifuge tubes or a 96 tube microtiter array multichannel pipettors and centrifuge adaptor for multiwell plates 20 505 Lyticase Solution 5 units ul in TE buffer s
95. med to 60 C to reliquefy 400 ul Recipe above 50 ul 20 ul PMSF stock solution 100X 20 ul Prechilled recipe above 30 ul Clontech Laboratories Inc www clontech com Protocol No PT3024 1 58 Version No PR742227 Yeast Protocols Handbook APPENDIX D Solution Formulations continued B For Transformation of Yeast Herring testes carrier DNA 10 mg ml Sonicated herring testes carrier DNA in solution can be purchased separately Cat No 630440 see Chapter XI for ordering information or can be prepared using a standard method Sambrook et al 1989 Just prior to use denature the carrier DNA by placing it in a boiling water bath for 20 min and immediately cooling it on ice Use only high quality carrier DNA nicked calf thymus DNA is not recommended PEG LiAc solution polyethylene glycol lithium acetate Prepare fresh just prior to use Final Conc To prepare 10 ml of solution PEG 4000 40 8 ml of 50 PEG TE buffer 1X 1 ml of 10X TE LiAc 1X 1 ml of 10X LiAc e Stock solutions 50 PEG 3350 Polyethylene glycol avg mol wt 3 350 Sigma Cat No P 3640 prepare with sterile deionized H O if necessary warm solution to 50 C to help the PEG go into solution 100 DMSO Dimethyl sulfoxide Sigma Cat No D 8779 10X TE buffer 0 1 M Tris HCI 10 mM EDTA pH 7 5 Autoclave 10X LiAc 1 M lithium acetate Sigma Cat No L 6883 Adjust to pH 7 5 with dilute acetic acid and autoclave C For galactosi
96. n pGADT7 pGAD GH pLexA and pAS2 1 during logarithmic growth of the yeast host cells Transcription is repressed in late log phase by the ethanol that accumulates in the medium as a by product of yeast metabolism Several Matchmaker cloning vectors contain a truncated 410 bp ADH7 promoter Table Il At one point itwas believed that only this portion was necessary for high level expression Beier amp Young 1982 In most vector constructs however this truncated promoter leads to low or very low levels of fusion protein expression Ruohonen etal 1991 Ruohonen etal 1995 Tornow amp Santangelo 1990 This observation has been confirmed at Clontech by quantitativeWestern blots unpublished data The high level expression reported by Beier amp Young 1982 was apparently due to a segment of DNA derived from pBR322 which was later found to coincidentally enhance transcriptional activity in yeast Tornow amp Santangelo 1990 In the Matchmaker vector pACT2 strong constitutive fusion protein expression is driven by the 410 bp truncated ADH7 promoter adjacent to this enhancing pBR322 segment The DNA BD cloning vector pGBKT7 used in MatchmakerTwo Hybrid System 3 contains a 700 bp fragment of the ADH7 promoter This trucated promoter leads to high level expression but no ethanol repression Ruohonen et al 1991 Ruohonen et al 1995 The AD cloning vector pB42AD and the alternative DNA BD vector pGilda used in the Matchmaker LexA Two Hybri
97. nd 0 1 mg of herring testes carrier DNA to a fresh 1 5 ml tube and mix Notes e For simultaneous cotransformation using two different plasmids use 0 1 ug of each plasmid an approximately equal molar ratio in addition to the 0 1 mg of carrier DNA e For transformations to integrate a reporter vector use at least 1 ug of linearized plasmid DNA in addition to the carrier DNA 13 Add 0 1 ml of yeast competent cells to each tube and mix well by vortexing 14 Add 0 6 ml of sterile PEG LiAc solution to each tube and vortex at high speed for 10 sec to mix 15 Incubate at 30 C for 30 min with shaking at 200 rpm 16 Add 70 ul of DMSO Mix well by gentle inversion Do not vortex 17 Heat shock for 15 min in a 42 C water bath 18 Chill cells on ice for 1 2 min 19 Centrifuge cells for 5 sec at 14 000 rpm at room temperature Remove the supernatant 20 Resuspend cells in 0 5 ml of sterile 1X TE buffer 21 Plate 100 ul on each SD agar plate that will select for the desired transformants To ensure that you will obtain a plate with well separated colonies also spread 100 ul of a 1 1000 1 100 and 1 10 dilution on 100 mm SD agar plates These will also provide controls for co transformation efficiency Note If you are performing a cotransformation plate controls to check transformation efficiency and markers of each plasmid On separate 100 mm plates spread 1 ul diluted in 100 ul H O on medium that will select for a single type
98. ndix D F 1X NaOAc Buffer Appendix D F Assay Buffer Appendix D F 1 5 ml cuvettes or 96 well flat bottom microtiter plates for OD measurements Preparation of Samples 1 Inoculate 2 5 ml of liquid synthetic dropout SD medium containing the appropriate dropout supplements with a yeast colony expressing the pair of proteins being analyzed It is advisable to set up triplicate cultures for each type of yeast colony being analyzed Fresh one to three week old colonies will give best results for liquid culture inoculation A single colony may be used for the inoculation if it is 2 3 mm in diameter Scrape the entire colony into the medium If the colonies on the master plate are smaller than 2 mm transfer several colonies into the medium Examples e For AH109 and Y190 cotransformants expressing interacting pairs of GAL4 BD and AD fusion proteins inoculate into SD His Leu Trp e ForAH109 andY190 cotransformants expressing non interacting pairs of fusion protein constructs inoculate into SD Leu Trp ForY187 tranformants expressing interacting or non interacting proteins inoculate into SD Leu Trp For non transformed AH109 Y187 and Y190 host strains inoculate into SD Ura 2 Incubate at 30 C overnight 16 18 hr with shaking 250 rpm 3 Vortex the cell culture tube for 0 5 1 min to disperse cell clumps then transfer 1 ml of the suspension to a clean cuvette and record the OD For accuracy the
99. nsformation efficiency is determined by the number of colonies growing on SD medium that selects for both plasmids and should be 10 cfu ug DNA Simultaneous cotransformation is generally preferred because it is simpler than sequential transformation and because of the riskthat expression of proteins encoded by the first plasmid may be toxic to the cells If the expressed protein is toxic clones arising from spontaneous deletions inthe first plasmid will have a growth advantage and will accumulate at the expense of clones containing intact plasmids However if there is no selective disadvantage to cells expressing the first cloned protein sequential transformation may be preferred because it uses significantly less plasmid DNA than simultaneous cotransformation In some cases such as when one of the two plasmids is the same for several different cotransformations sequential transformations may be more convenient Scaling up or down The small scale yeast transformation procedure described here can be used for upto 15 parallel transformations and uses 0 1 ug of each type of plasmid Depending on the application the basic yeast transformation method can be scaled up without a decrease in transformation efficiency If you plan to perform a two hybrid library screening you will need a large or library scale transformation procedure which will require significantly more plasmid DNA Please refer to your Matchmaker system specific User Manual for f
100. nt claims outside the US 5 079 352 and 6 127 155 The purchase of this product includes a limited non transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser s own internal research No right under any other patent claim such as method claims in U S Patents Nos 5 210 015 5 487 972 5 994 056 and 6 171 785 and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby conveyed by the purchase of this product expressly by implication or by estoppel This product is for research use only Diagnostic uses require a separate license from Roche Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA Clontech the Clontech logo and all other trademarks are the property of Clontech Laboratories Inc unless noted otherwise Clontech is aTakara Bio Company 2008 Clontech Laboratories Inc Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 3 Yeast Protocols Handbook I Introduction The Yeast Protocols Handbook provides background information and general yeast protocols that complement oursystem specific User Manuals The protocols inthis Handbook have been optimized with our yeast based Matc
101. ny lift Filter Assay Reagents and Materials Required e Whatman No 5 or VWR Grade 410 paper filters sterile Notes 75 mm filters e g VVVR Cat No 28321 055 can be used with 100 mm plates 125 mm filters e g VVVR Cat No 28321 113 can be used with 150 mm plates e Alternatively 85 and 135 mm filters can be specially ordered from Whatman e Nitrocellulose filters also can be used but they are prone to crack when frozen e Forceps for handling the filters e Z buffer Appendix D e Z buffer X gal solution Appendix D e X gal stock solution Appendix D e Liquid nitrogen 1 For best results use fresh colonies i e grown at 30 C for 2 4 days 1 3 mm in diameter Notes If only a few colonies are to be assayed streak them or spread them in small patches directly onto master SD selection agar plates Incubate the plates at 30 C for an additional 1 2 days and then proceed with the P galactosidase assay below e Use the SD selection medium appropriate for your system and plasmids When testing LexA transformants be sure to use gal raff induction medium 2 Prepare Z buffer X gal solution as described in Appendix D 3 For each plate of transformants to be assayed presoak a sterile Whatman No 5 or VVVR grade 410 filter by placing it in 2 5 5 ml of Z buffer X gal solution in a clean 100 or 150 mm plate Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Vi Yeast
102. o PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 Yeast Protocols Handbook APPENDIX D Solution Formulations continued For TCA Protein Extraction Method e 20 w vTCA in H O Store at 4 C See Sambrook et al 1989 for tips on preparing TCA solutions e TCA Buffer Place on ice to prechill before use Add the protease inhibitor solution and pMSF immediately prior to use Tris HCl pH 8 20 mM Ammonium acetate 50 mM EDTA 2 mM Deionized H O 9 7 ml Protease inhibitor solution 50 ul ml PMSF SDS glycerol stock solution SDS 73 wiv Glycerol 29 1 viv Tris base 83 3 mM Bromophenol blue Deionized H O Tris EDTA solution Tris base 200 mM EDTA 20 mM Deionized H O TCA Laemmli loading buffer Prepare fresh just prior to use SDS glycerol stock solution Tris EDTA solution B mercaptoethanol PMSF Protease inhibitor solution Deionized H O To prepare 10 ml of TCA buffer 200 ul of a 1MM stock solution 66 6 ul of a 7 5 M stock solution 40 ul of a 0 5 M stock solution 500 ul prechilled recipe above 100 ul of 100X stock solution To prepare 12 ml 3 5 ml of a 2596 stock solution 3 5 ml of 10096 1 0 ml of a 1 M stock solution not pH adiusted Spatula tip full To a final volume of 12 ml To prepare 10 ml 2 0 ml of 1 M stock solution not pH adiusted 0 4 ml of a 0 5 M stock solution 7 6 ml To prepare 1 ml 480 ul Stocksolution may need to be war
103. o disperse the cells For each type of control plasmid to be used place several transformant colonies in 3 ml of YPD in a sterile 10 ml conical tube Vigorously vortex the tube to disperse the cells Aliquot 20 ul of the cell suspension from Step 3 b into each well of a vertical column Use a separate column for each plasmid of interest to be tested Aliquot 20 ul of the cell suspension from Step 3 c into each well of a horizontal row Use a separate row for each type of control plasmid Place plate on a rotating platform shaker and incubate at 30 C for 6 18 hr at 200 rpm Note Do not rotate at a higher speed or the medium will spill out of the wells Spread 100 ul of each mating culture on 100 mm plates containing the appropriate SD minimal medium and proceed to next step 4 Incubate plates at 30 C for 3 5 days to allow diploid cells to form visible colonies o Score for growth on the SD agar plates 6 Confirm nutritional and reporter phenotypes of diploids To detect or reconfirm protein protein interactions assay the fresh diploid colonies from the SD selection plates Step 4 above for B gal activity using the colony lift filter assay SectionVI C Discard any B gal positive colonies that contain the candidate library plasmid alone Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 Yeast Protocols Handbook X References Yeast Two Hybrid System References Bartel P L
104. o reduce variability in liquid assays assay five separate transformant colonies and perform each assay in triplicate e t is important that the colonies to be assayed for a and galactosidase activity are grovving on the appropriate SD minimal medium SD dropout medium is used to keep selective pressure on the hybrid plasmids and in the case of the Matchmaker LexA Two Hybrid System the acZ reporter plasmid up to the time the cells are lysed for the assay The type of SD medium needed depends on the plasmids and host strains used Furthermore when working with a acZ reporter under the control of the inducible GAL7 promoter such as in the LexA System the SD medium must contain galactose not glucose as the carbon source See the system specific User Manual for media recommendations B Galactosidase Assays X gal must be used as the f galactosidase substrate for solid support assays because of its high degree of sensitivity X gal is 10 fold more sensitive than ONPG Although more sensitive than X gal Galacton Star is not recommended for agar plate and filter assays because it gives troublesome background The filter and liquid B galactosidase assays described here use at least one freeze thaw cycle in liquid nitrogen to lyse the yeast cell walls Freeze thaw cycles are a rapid and effective cell lysis method which permits accurate quantification of B galactosidase activity Schneider et al 1996 The colony lift filter
105. of AH109 PJ69 2A HF7c and CG 1945 and the JacZ reporter of Y190 Y187 and SFY526 are all tightly regulated by the intact GAL7 promoter including the GAL7 UAS and GAL7 minimal promoter In HF7c and CG1945 lacZexpression is under control of VAS 17 mery g and the extremely weak minimal promoter of the yeast cytochrome C1 CYC1 gene lacZ under the control of the intact GAL7 promoter can be expressed at 10X the level obtained with the VAS 6 17 mer xa CYC7 minimal promoter construct under similar induction conditons Clontech Laboratories unpublished data Therefore some weak or transient two hybrid interactions may not be detectable in HF7c or CG1945 unless you use a highly sensitive B galactosidase assay such a liquid culture assay using a chemiluminescent substrate Chapter VI F The ADE2 reporter of PJ69 2A and AH109 is tightly regulated by the intact GAL2 promoter whose induction properties are similar to those of the GAL7 promoter In AH109 2 15 under the control of the MEL1 UAS and minimal promoter The MEL1 promoter is stonger than the UAS 17 mer xa CYCT1 minimal promoter but weaker than the GAL7 promoter Aho et al 1997 Reporter genes under the control of a minimal HIS3 promoter The native yeast HIS3 promoter contains a UAS site recognized by the transcriptional activator GCN4 and twoTATA boxes GCN4 regulates one of the TATA boxes TR while the other TATA box TC drives low level constitutive expression of H S3 lyer
106. of colonies to be screened presoak aWhatman 3 MM paper in the sorbitol EDTA DTT solution 4 Using forceps place a sterile prelabeled dry nylon membrane over the surface of the plate of colonies to be assayed Gently rub the membrane with the side of the forceps to help colonies cling to the membrane 5 Poke holes through the membrane into the agar in three or more assymetric locations to orient the membrane to the master plate 6 When the membrane has been evenly wetted carefully lift it off the agar plate with forceps and allow it to air dry briefly 5 min Place membrane colony side up on a presoaked sheet of Whatman 3 MM paper from Step 3 above and incubate for 30 min 7 Optional Place membranes at 70 C for 5 min then thaw at room temperature for one or more cycles to facilitate the disruption of the cell walls 8 Dilute the B glucuronidase 1 500 in sorbitol EDTA Use 2 ul of the 100 000 units ml 8 glucuronidase stock per ml of sorbitol EDTA to give a final concentration of 200 units ml Allow 3 5 ml of diluted B glucuronidase per filter to be screened 9 For each membrane to be screened cut another piece of Whatman 3 MM paper to fit inside a 100 mm petri dish Place the paper disc in the dish containing the diluted B glucuronidase to saturate the paper Remove excess liquid 10 Carefully layer the nylon membrane colony side up ontop of the B glucuronidase soaked filter Avoid trapping air bubble
107. of plasmid 22 Incubate plates up side down at 30 C until colonies appear generally 2 4 days 23 To calculate the cotransformation efficiency count the colonies cfu growing on the dilution plate from Step 22 above that has 30 300 cfu cfu x total suspension vol ul cfu ug DNA Vol plated ul x dilution factor x amt DNA used ug In a cotransformation this is the amount of one of the plasmid types not the sum of them If you have used unequal amounts of two plasmids use the amount of the lesser of the two Sample calculation e 100 colonies grew on the 1 100 dilution plate dilution factor 0 01 plating volume 100 ul e resuspension volume 0 5 ml e amount of limiting plasmid 0 1 ug 100 cfu x 0 5ml x 10 ul ml 5x 105 cfu ug DNA 100 ul x 0 01 x 0 1 ug 24 Pick the largest colonies and restreak them on the same selection medium for master plates Seal plates with Parafilm and store at 4 C for 3 4 weeks Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 21 Yeast Protocols Handbook V Yeast Transformation Procedures continued F Troubleshooting Yeast Transformation The overall transformation efficiency should be at least 10 cfu ug for transformation with a single type of plasmid and 103 cfu ug for simultaneous cotransformation with two types of plasmids If your cotransformation efficiency is lower than expected calculate the transformation effic
108. ology Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Printen J A amp Sprague G F Jr 1994 Protein interactions in the yeast pheromone response pathway Ste5p interacts with all members of the MAP kinase cascade Genetics 138 609 619 Roux K H 1995 Optimization and troubleshooting in PCR PCR Methos Appl 4 5185 5194 Ruohonen L Aalto M K amp Ker nen S 1995 Modifications to the ADH7 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins Journal of Biotechnology 39 193 203 Ruohonen L Penttila M amp Ker nen S 1991 Optimization of Bacillus a amylase Production by Saccharomyces cerevisiae Yeast 7 337 346 Sambrook J Fritsch E F 8 Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Sadowski l Niedbala D Wood K amp Ptashne M 1991 GAL4 is phosphorylated as a consequence of transcriptional activation Proc Natl Acad Sci USA 88 10510 10514 Schiestl R H amp Gietz R D 1989 High efficiency transformation of intact cells using single stranded nucleic acids as a carrier Curr Genet 16 339 346 Schneider S Buchert M amp Hovens C M 1996 An in vitro assay of B galactosidase from yeast BioTechniques 20 960 962 Smirnov M N Smirnov V N Budovvsky E l Inge Vechtomov S G amp Serebrjakov N G 1967 Red pigment of adenine deficient yeast Sacc
109. ompetent Cells Miscellaneous related reagents X a Gal Luminescent B gal Detection Kit Il Advantage 2 PCR Kit Advantage 2 Polymerase Mix CHROMA SPIN TE 400 Columns Protocol No PT3024 1 www clontech com Version No PR742227 630439 630440 630441 630409 630410 630411 630412 630420 630421 many 630301 630302 630303 630449 631604 many many 630433 630402 5399 1 630403 630442 630406 630405 630404 many many 630435 630434 630407 631712 639206 639207 639201 639202 636076 Clontech Laboratories Inc 49 Yeast Protocols Handbook APPENDIX A Glossary of Technical Terms Note Many of these terms have other meanings in different contexts For brevity we have included only definitions relevant to this Yeast Protocols Handbook allele One of two or more forms that can exist at a given genetic locus e g his3 200 is a mutant allele and H S3 is a wild type allele at the his3 locus In standard yeast nomenclature mutant alleles are written in lower case italics while wild type alleles are written in upper case italics auxotroph A strain of yeast or other microorganisms that will proliferate only when the medium is supplemented with some specific nutrient not normally required by the organism For example Trp yeast strains are auxotrophic for tryptophan Trp they require Trp in the medium cis acting element or cis acting locus A DNA sequence that affects the transcriptional activi
110. on and blank tubes Do not add Z buffer prior to freezing samples 14 Start timer Immediately add 160 ul of ONPG in Z buffer to the reaction and blank tubes 15 Place tubes in a 30 C incubator 16 After the yellow color develops add 0 4 ml of 1 M Na CO to the reaction and blank tubes Record elapsed time in minutes Notes Thetime needed will vary 3 15 min for the single plasmid B gal positive control 30 min for a two hybrid positive control and up to 24 hr for weaker interactions e The yellow color is not stable and will become more intense with time You will need to run a new blank tube with every batch 17 Centrifuge reaction tubes for 10 min at 14 000 rpm to pellet cell debris 18 Carefully transfer supernatants to clean cuvettes Note The cellular debris if transferred with the supernatant will strongly interfere with the accuracy of this test 19 Calibrate the spectrophotometer against the blank atA and measure the OD ofthe samples relative to the blank The ODs should be between 0 02 1 0 to be within the linear range of the assay 20 Calculate B galactosidase units 1 unit of B galactosidase is defined as the amount which hydrolyzes 1 umol of ONPG to o nitrophenol and D galactose per min per cell Miller 1972 Miller 1992 B galactosidase units 1 000 x OD t x V x where t elapsed time in min of incubation V ml x concentration factor Asoo Of 1 ml
111. on when it is appropriate to use this procedure To promote integration of yeast plasmids follow the small scale LiAc transformation procedure Section V E below with the following exceptions Before transformation linearize 1 4 ug of the reporter vector by digesting it with an appropriate restriction enzyme in a total volume of 40 ul at 37 C for 2 hr Electrophorese a 2 ul sample of the digest on a 1 agarose gel to confirm that the plasmid has been efficiently linearized Notes If the vector contains a yeast origin of replication i e 2 u ori it will be necessary to remove it before you attempt to integrate the vector Thevector should be linearized within the gene encoding the transformation i e nutritional selection marker However if the digestion site is within a region that is deleted in the host strain the plasmid will not be able to integrate Please refer to your product specific User Manual for recommended linearization sites At Step 12 add 1 4 ug of the linearized reporter plasmid 100 ug of carrier DNA for each reporter plasmid also set up a control transformation with undigested plasmid 100 ug carrier DNA At Step 20 resuspend cells in 150 ul of TE buffer Plate the entire transformation mixture on one plate of the appropriate SD medium to select for colonies with an integrated reporter gene E Small scale LiAc Yeast Transformation Procedure 1 O N Inoculate 1 ml ofYPD or S
112. ose and glucose The a Gal Quantitative Assay allows you to specifically identify and measure this catalytic activity using p nitrophenyl a p galactoside PNP a Gal a colorless compound that yields a yellow product p nitrophenol upon hydrolysis The a Gal Quantitative Assay can be used to measure the extracellular a galactosidase activity produced during the culture of any yeast strain that carries the MEL1 gene MELTis endogenousto many butnotall yeast strains Liljestr m 1985 Post Beittenmiller etal 1984 Table IX provides alist of the GAL4 based Matchmaker yeast strains known to carrythe MEL1 gene This list includes strain AH109 used in Clontech s MatchmakerTwo Hybrid System 3 Cat No 630303 and included with Clontech s GAL4 based cDNA libraries MEL1 has been shown to be a sensitive in vivo reporter for GAL4 based two hybrid assays Aho et al 1997 The quantitative nature of the a Gal Assay makes it possible to compare the degree of MEL1 expression in different Matchmaker two hybrid host cell populations containing different pairs of interacting proteins or to measure differences in the relative strength of binding between mutant forms of interacting proteins Principle of the o Gal Quantitative Assay In the a Gal Quantitative Assay the catalytic activity of a galactosidase is monitored colorimetrically by measuring the rate of hydrolysis of the chromogenic substrate p nitrophenyl a p galactoside PNP a Gal One of the p
113. ose as the carbon sources instead of glucose If you are not using Clontech s SD Gal Raf Minimal Base be sure to obtain high quality galactose that is not contaminated by glucose Stock solutions for use with SD Media 1M 3 AT 3 amino 1 2 4 triazole Sigma Cat No A 8056 prepare in deionized H O and filter sterilize Store at 4 C Store plates containing 3 AT sleeved at 4 C for up to 2 months e 10X BU Salts Dissolve the following components in 1 L total of H O 70 g Na HPO 7H O 30 g NaH PO Adjust to pH 7 then autoclave and store at room temperature e Carbon sources filter sterilized or autoclaved Note Autoclave at 121 C for 15 min autoclaving at a higher temperature for a longer period of time or repeatedly may cause the sugar solution to darken and will decrease the performance of the medium e 40 Dextrose glucose Store at 4 C e 40 Galactose for LexA Two Hybrid System D Galactose e g Sigma Cat No G 0750 Store at 4 C e 40 Raffinose for LexA Two Hybrid System Store at 4 C 1 mg ml 1000X CHX Cycloheximide Sigma Cat No C 7698 prepare in deionized H O filter sterilize and aliquot Store at 4 C for up to 2 months Store plates containing CHX sleeved at 4 C for up to 1 month 50 mg ml kan kanamycin prepare in deionized H O filter sterilize and aliquot Store at 20 C for up to 1 month Store plates containing kan sleeved at 4 C for up to 1 month Clontech Laboratories Inc www
114. ould add aspartic acid to the 10X DO Solution a 10X solution of aspartic acid is 1000 mg L 10X dropout supplements may be autoclaved and stored at 4 C for up to 1 year Nutrient 10X Concentration Sigma Cat No L Adenine hemisulfate salt 200 mg L A 9126 L Arginine HCI 200 mg L A 5131 L Histidine HCI monohydrate 200 mg L H 8125 L soleucine 300 mg L l 2752 L Leucine 1000 mg L L 8000 L Lysine HCI 300 mg L L 5626 L Methionine 200 mg L M 9625 L Phenylalanine 500 mg L P 2126 L Threonine 2000 mg L T 8625 L Tryptophan 200 mg L T 0254 L Tyrosine 300 mg L T 3754 L Uracil 200 mg L U 0750 L Valine 1500 mg L V 0500 Example To make one liter of 10X Leu Trp DO Solution combine the following e 200 mg adenine hemisulfate 200mg arginine HCl e 200 mg histidine HCI monohydrate 300 mg _ isoleucine 300 mg lysine HCl 200mg methionine e 500mg phenylalanine e 2000 mg threonine e 300mg tyrosine e 200mg uracil 1500 mg _ valine 1L Dissolve components in 1 L deionized H O Autoclave Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 Yeast Protocols Handbook APPENDIX C Media Recipes continued B E coliMEDIA e Hanahan s SOC Medium Final concentration To Prepare One Liter Bactotryptone 2 20 g Yeast extract 0 5 NaCl 10 mM 10 ml of 1 M NaCl KCI 2 5 mM 2 5 ml of 1 M KCI MgCl 10 mM 10 ml of 1 M MgCl 6 H O MgSO 10 mM 10 ml of 1 M MgSO 7H O Glucose
115. r Biology by Guthrie amp Fink 1991 No V2010 1 Molecular Biology and Genetic Engineering of Yeasts edited by Heslot amp Gaillardin 1992 Stargell amp Struhl 1996 and Pringle et al 1997 No V2365 1 UAS andTATA regions are basic building blocks of yeast promoters The initiation of gene transcription in yeast as in other organisms is achieved by several molecular mechanisms working in concert All yeast structural genes i e those transcribed by RNA polymerase II are preceded by a region containing a loosely conserved sequence TATA box that determines the transcription start site and is also a primary determinant of the basal transcription level Many genes are also associated with cis acting elements DNA sequences to which transcription factors and other trans acting regulatory proteins bind and affect transcription levels The term promoter usually refers to both the TATA box and the associated cis regulatory elements This usage is especially common when speaking of yeast gene regulation because the cis regulatory elements are relatively closely associated with the TATA box Yoccum 1987 This is in contrast to multicellular eukaryotes where cis regulatory elements such as enhancers can be found very far upstream or downstream from the promoters they regulate In this text minimal promoter will refer specifically to the TATA region exclusive of other cis acting elements The minimal promoter or TATA box
116. roducts of this reaction p nitrophenol displays a strong absorption band at 410 nm o galactosidase PNP a Gal H20 p nitrophenol p galactose Amax 410 nm Because yeast naturally secrete a galactosidase into the surrounding medium it is more convenient to assay than galactosidase an intracellular enzyme encoded by the lacz reporter gene The a galactosidase assay is carried out by simply combining a small aliquot of cell free culture media with a fixed volume of Assay Buffer cell lysis is not necessary After a prescribed incubation time usually 60 min the absorbance at 410 nm OD which is proportional to moles p nitrophenol liberated is recorded and used to calculate the concentration of a galactosidase in milliunits ml x cell To monitor MEL1 expression directly on nutritional selection plates use X a Gal Cat No 630407 X a Gal can be included in the medium before pouring plates or spread on top of the medium before plating liquid cultures As a galactosidase accumulates in the medium it hydrolyzes X a Gal causing yeast colonies to turn blue Instructions for preparing X a Gal indicator plates are given in the X a Gal Protocol at a Glance PT3353 2 supplied with each purchase of the substrate Directions for use can also be downloaded from our website at www clontech com Clontech Laboratories Inc www clontech com Protocol No PT3024 1 24 Version No PR742227 Yeast Protocols Handbook VI a and p
117. rogram Consult your scintillation counter s manufacturer for further information about this software 25 For detection methods described in Steps 22 24 Calculate the B galactosidase activity in terms of RLU OD unit of cell culture Note that Miller unit calculations are not possible using these methods 26 Optional If you have set up B galactosidase standards prepare a standard curve of RLU vs the amount of B galactosidase Estimate the quantity of B galactosidase inthe unknown samples using the standard curve Determine the amount of enzyme per OD unit of cell culture The final units of cells assayed per sample is calculated as follows OD oo from Step 5 x vol from Step 18 x conc factor from Step 9 27 Detection by exposure of x ray film Light emission can also be recorded by exposure of x ray film to reaction samples in opaque 96 vvell flat bottom microtiter plates The relative intensity of the resulting spots on the film can be estimated by comparison to positive and negative controls Note that x ray film is several orders of magnitude less sensitive than aluminometer or scintillation counter Overlay the microtiter plate with x ray film cover the film with plastic wrap and place a heavy object such as a book on top to hold the film in place Expose the film at room temperature for 5 30 min Note To compare samples accurately they must be within the linear response capability of the x ray film We there
118. rtex vigorously for 1 min e Centrifuge at 14 000 rpm for 5 min 22 First supernatant e Place on ice e Boil for 3 5 min e Vortex vigorously for 1 min e Centrifuge at 14 000 rpm for for 5 min Pellet discard Second Combine with supernatant second supernatant e Place on ice Combined supernatants e Immediately load gel or freeze at 70 C or colder Figure 2 Urea SDS protein extraction method Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 IV Yeast Protocols Handbook Preparation of Yeast Protein Extracts continued D Preparation of Protein Extracts TCA Method Figure 3 Horecka J personal communication Reagents and Materials Required 1 5 ml screw cap microcentrifuge tubes Glass beads 425 600 um Sigma Cat No G 8772 Protease inhibitor solution Appendix D A PMSF Stock solution Appendix D A Add as necessary throughout the protocol Recommended Bead Beater BioSpec Bartlesville OK Note If you do not have access to a Bead Beater a high speed vortexer can be used instead However vortexing is not as effective as bead beating at disrupting the cells TCA buffer Appendix D A ce cold 20 w vTCA in H O see Sambrook et al 1989 for tips on preparingTCA solutions TCA Laemmli loading buffer Appendix D A Note Unless otherwise stated keep protein samples on ice ___ 1 Thaw cell pellets on ice 1
119. s Inc Ch 20 0 and 20 1 Guthrie C amp Fink G R 1991 Guide to yeast genetics and molecular biology In Methods in Enzymology Academic Press San Diego 194 1 932 Harper J W Adami G R Wei N Keyomarsi K amp Elledge S J 1993 The p21 Cdk interacting protein Cip1 is a potent inhibitor of G1 cyclin dependent kinases Cell 75 805 816 Heslot H amp Gaillardin C eds 1992 Molecular Biology and Genetic Engineering of Yeasts CRC Press Inc Hill J Donald K A amp Griffiths D E 1991 DMSO enhanced whole cell yeast transformation Nucleic Acids Res 19 5791 Hope 1 A 8 Struhl K 1986 Functional dissection of a eukaryotic transcriptional protein GNN4 of yeast Cell 46 885 894 H Fukada Y Murata K amp Kimura A 1983 Transformation of intact yeast cells treated with alkali cations J Bacteriol 153 163 168 vvabuchi K Li B Bartel P amp Fields S 1993 Use of the two hybrid system to identify the domain of p53 involved in oligomerization Oncogene 8 1693 1696 lyer V amp Struhl K 1995 Mechanism of differential utilization of the his3 TR and TC TATA elements Mol Cell Biol 15 7059 7066 James P Halladay J amp Craig E A 1996 Genomic libraries and a host strain designed for highly efficient two hybrid selection in yeast Genetics 144 1425 1436 Johnston M Flick J S 8 Pexton T 1994 Multiple mechanisms provide rapid and stringent gluco
120. s and sequencing primers and yeast media including Minimal SD Base and many different formulations of Dropout DO Supplement Finally the pHA CMV and pMyc CMV Vector Set Cat No 631604 can be used to confirm protein interactions in mammalian cells For ordering information on these products please see Chapter XI of this Handbook or the Clontech Catalog Clontech Laboratories Inc www clontech com Protocol No PT3024 1 Version No PR742227 Yeast Protocols Handbook ll Introduction to Yeast Promoters Yeast promoters and other cis acting regulatory elements play a crucial role in yeast based expression systems and transcriptional assays such as the Matchmaker One and Two Hybrid Systems Differences in the promoter region of reporter gene constructs can significantly affect their ability to respond to the DNA binding domain of specific transcriptional activators promoter constructs also affect the level of background or leakiness of gene expression and the level of induced expression Furthermore differences in cloning vector promoters determine the level of protein expression and in some cases confer the ability to be regulated by a nutrient such as galactose in the case of the GAL7 promoter This chapter provides a brief introduction to several commonly used yeast promoters and cis regulatory elements For further information on the regulation of gene expression in yeast we recommend the Guide to Yeast Genetics and Molecula
121. s in between the two layers Cover the dish Incubate the membrane on the filter for up to 6 hr at 37 C until gt 80 of the cells lack a cell wall Clontech Laboratories Inc www clontech com Protocol No PT3024 1 42 Version No PR742227 Yeast Protocols Handbook IX Additional Useful Protocols continued Note The extent of cell wall removal can be determined by removing a small quantity of cells from the filter to a drop of sorbitol EDTA on a microscope slide and observing directly with a phase contrast microscope at 260X magnification Cells lacking a cell wall are nonrefractile 11 Place membrane on Whatman 3 MM paper saturated with 0 5 M NaOH for 8 10 min 12 Place membrane on Whatman 3 MM paper saturated with 0 5 M Tris HCI pH 7 5 6 SSC for 5 min Repeat step 12 with a second sheet of presoaked Whatman 3 MM paper 13 Place membrane on Whatman 3 MM paper saturated with 2X SSC for 5 min Then place membrane on dry Whatman paper to air dry for 10 min 14 Bake membrane at 80 C for 90 min in a vacuum oven or UV cross link 15 Proceed as for bacterial filter hybridization Ausubel et al 1994 B Generating Yeast Plasmid Segregants For some applications it is useful to generate a segregant strain that has only a single type of plasmid from yeast cotransformants containing more than one kind of plasmid There are several ways this can be accomplished The most reliable but also most time consuming way is to isolate t
122. se d s y u uoiss idx S H Aye 7 Toux Aq n ou si ugsqv5d p 015 BUS Buis e ABU YoIYyM JO ypes 170e7d pue ISIHd Wo Way sinBunsip o lqissod 1 sayew ews Aq 1nd JOU 1e an gegd pue sjHEgd 5 enuey 4 sn uu 1s s plqAH uQ JexeWY e 94 u p piAold s u nb s SOM BUS Buliuo 5 pue sdew uon ns d UOI ELUIJOJU 40199A EUOH PPV q suonoe1 1ul u 1o id vNGdG ll ds Bu 119581 p 40 ulesjs 158 AlEss v y o p sn Udy S 0ni1suoo u spliuuise d 1 yod y uo JO S H 24 OJU p vol 1snui u nb s vNd 2618 u nb s uoniuBoo i Anend e WajsAs piuq u uo Y l e due Svan v0 0 7779 ul l 38612 9661 7 19 ON 04 do33 69 en JO O1JUOD Zoe Izoeqd due 01 Yt u uu 6661 8 29 04 do33 ys SIH JO 104u02 ABPUN SIH L ISIHd due evyn OL Z G 1 8661 8 29 04 402937 89 SIH ean JO ojluoo 1 punN SIH ISIHd 97 due 7997 av pq 9661 Je 19 onq pl H097 9z n o pasny eed uunuu uigsqv d due vun 0 9 1zoeqd ul seus Bulpulq 200 9661 8 19 ON l OYX 023 v9 eun JO o13uoo s pun 228 anigesd dwe EVYN SIH O L 4 ISIHd ul says Buipuiq ggd 96
123. se repression of GAL gene expression in Saccharomyces cerevisiae Mol Cell Biol 14 3834 3841 Kaiser P amp Auer B 1993 Rapid shuttle plasmid preparation from yeast cells by transfer to E coli BioTechniques 14 552 Kellogg D E Rybalkin l Chen S Mukhamedova N Vlasik T Seibert P amp Chencik A 1994 TaqStart Antibody Hot start PCR facilitated by a neutrualizing monoclonal antibody directed against Taq DNA polymerase BioTechniques 16 1134 1137 Lazo P S Ochoa A G amp Gascon S 1978 a galactosidase melibiase from Saccharomyces Carlsbergenesis Structural and kinetic properties Arch Biochem Biophys 191 316 324 Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 47 Yeast Protocols Handbook X References continued Li B amp Fields S 1993 Identification of mutations in p53 that affect its binding to SV40T antigen by using the yeast two hybrid system FASEB J 7 957 963 Li L Elledge S J Peterson C A Bales E S amp Legerski R J 1994 Specific association between the human DNA repair proteins XPA and ERCC1 Proc Natl Acad Sci USA 91 5012 5016 Liljestr m P L 1985 The nucleotide sequence of the yeast MEL1 gene Nucleic Acids Res 13 7257 7268 Ling M Merante F amp Robinson B H 1995 A rapid and reliable DNA preparation method for screening a large number of yeast clones by polymerase chain reaction Nuclei
124. sformation controls When setting up any type of transformation experiment be sure to include proper controls for transformation efficiencies In the case of simultaneous cotransformation it is important to determine the transformation efficiencies of both plasmids together as well as of each type of plasmid independently That way if the cotransformation efficiency is low you may be able to determine whether one of the plasmid types was responsible seeTroubleshooting Guide Section F Therefore be sure to plate an aliquot of the transformation mixture on the appropriate SD media that will select for only one type of plasmid Example calculations are shown in Section V E When screening a library or performing a one or two hybrid assay you will need additional controls as explained your system specific User Manual B Reagents and Materials Required Note The Yeastmaker Yeast Transformation System Cat No 630439 contains all the solutions except media H O and DMSO required for yeast transformation Yeastmaker reagents have been optimized for use in the Matchmaker One and Two Hybrid Systems YPD or the appropriate SD liquid medium Sterile 1X TE 1X LiAc Prepare immediately prior to use from 10X stocks stock recipes in Appendix D B Sterile 1 5 ml microcentrifuge tubes for the transformation e Appropriate SD agar plates 100 mm diameter Notes Prepare the selection media and pour the required number of agar plates in advanc
125. strains and later is controlled by including 3 aminotriazole in the medium to suppress background growth Reporter genes under the control of LexA operators In LexA based two hybrid systems the DNA BD is provided by the entire prokaryotic LexA protein which normally functions as a repressor of SOS genes in E coli when it binds to LexA operators which are an integral part of the promoter Ebina et al 1983 When used in the yeast two hybrid system the LexA protein does not act as a repressor because the LexA operators are integrated upstream of the minimal promoter and coding region of the reporter genes LEU2 reporter expression in yeast strain EGY48 is under the control of six copies of the LexA operator op sequence and the minimal LEU2 promoter In the acZ reporter plasmids acZ expression is under control of 1 8 copies of the LexA op Estojak et al 1995 and the minimal GAL7 promoter Because all of the GAL7 UAS sequences have been removed from the acZ reporter plasmids West et al 1984 this promoter is not regulated by glucose or galactose Promoters used to drive fusion protein expression in two hybrid cloning vectors The ADH7 promoter or a truncated version of it is the promoter used to drive expression of the fusion proteins in most of the Matchmaker cloning vectors The 1500 bp full length ADH1 promoter Ammerer 1983 Vainio GenBank accession number Z25479 leads to high level expression of sequences under its control i
126. tain one or more conserved palindromic sequences to which the GAL4 protein binds Guthrie amp Fink 1991 Giniger et al 1985 reviewed in Heslot 8 Gaillardin 1992 The 17 mer consensus sequence referred to here as UAS 17 mevr functions in an additive fashion i e multiple sites lead to higher transcription levels than a single site Giniger amp Ptashne 1988 The protein binding sites of the GAL1 GAL2 MEL1 UASs and the VAS e Consensus Sequence are shown in Figure 1 The tight regulation of the GAL UASs by GAL4 makes it a valuable tool for manipulating expression of reporter genes in two hybrid systems that are dependent on the GAL4 DNA BD However in such systems the yeast host strains must carry deletions of the ga 4 and gal80 genes to avoid interference by endogenous GAL4 and GAL80 proteins thus no significant glucose repression is observed in these strains and no induction is observed unless a two hybrid interaction is occurring Therefore nutritional regulation of GAL UASs is not a feature of GAL4 based two hybrid systems However the host strain used in the LexA system does support galactose induction as it is wild type for GAL4 and GAL80 functions In the GAL4 based MatchmakerTwo Hybrid Systems either an intact GAL1 GAL2 or MEL1 UAS or an artifically constructed UAS consisting of three copies of the 17 mer consensus binding sequence is used to confer regulated expression on the reporter genes Table The H S3 reporter
127. that is already boiling If samples are placed in the water before it has reached boiling temperature a major yeast protease Proteinase B will be activated Proteinase B is a serine protease of the subtilisin family e Dephosphorylation of a normally phosphoryated fusion protein may have occurred during sample preparation Sodium fluoride NaF may be added to the protease inhibitor stock solution to help prevent dephosphorylation Sadowski et al 1991 3 If you are running a reducing gel make sure that the protein sample has been completely reduced with either dithiothreitol or 2 mercaptoethanol prior to loading the gel Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 17 Yeast Protocols Handbook V Yeast Transformation Procedures A General Information LiAc mediated yeast transformation There are several methods commonly used to introduce DNA into yeast including the spheroplast method electroporation and the lithium acetate LiAc mediated method reviewed in Guthrie amp Fink 1991 At Clontech we have found the LiAc method Ito et al 1983 as modified by Schiestl amp Gietz 1989 Hill et al 1991 and Gietz et al 1992 to be simple and highly reproducible This chapter provides detailed protocols for using the LiAc procedure in a standard plasmid transformation and ina modified transformation to integrate linear DNA into the yeast genome In the LiAc transformation me
128. the L29 protein of the yeast ribosome Cycloheximide a drug which blocks polypeptide elongation during translation prevents the growth of cells that contain the wild type CYH2 gene Cycloheximide resistance results from a single amino acid change in the CYH2 protein Cells containing both the sensitive wild type and the resistant mutant CYH2 alleles fail to grow on medium containing cycloheximide Therefore the loss of a CYH2 containing plasmid can be selected for directly if the host carries the resistant allele chromosomally Guthrie amp Fink 1991 pp 306 307 a From each of the restreaked Cyh5 cotransformants of interest pick a colony 1 3 mm in diameter and resuspend it in 200 ul of sterile H O Vortex thoroughly to disperse the cells Note Do not patch or streak cells from the colony over to the cycloheximide containing medium Cells transferred in this way are at too high a density for the cycloheximide selection to work b Spread 100 ul of the cell suspension onto an SD Leu cycloheximide plate Also spread 100 ul of a 1 100 dilution Note The concentration of eycloheximide to use in the medium depends on the hoststrain For example use 1 0 ug ml for CG 1945 10 0 ug ml forY190 c Incubate the plate at 30 C until individual Cyh colonies appear This usually takes 3 5 days d Transfer the Cyh colonies to appropriate SD selection plates to verify that they have lost the CYH 2 bearing plasmid and retained the plasmid of
129. thod yeast competent cells are prepared and suspended in a LiAc solution with the plasmid DNA to be transformed along with excess carrier DNA Polyethylene glycol PEG with the appropriate amount of LiAc is then added and the mixture of DNA and yeast is incubated at 30 C After the incubations DMSO is added and the cells are heat shocked which allows the DNA to enter the cells The cells are then plated on the appropriate medium to select for transformants containing the introduced plasmid s Because in yeast this selection is usually nutritional an appropriate synthetic dropout SD medium is used Simultaneous vs sequential transformations The LiAc method for preparing yeast competent cells typically results in transformation efficiencies of 10 per ug of DNA when using a single type of plasmid When the yeast is simultaneously cotransformed with two plasmids having different selection markers the efficiency is usually an order of magnitude lower due to the lower probability that a particular yeast cell will take up both plasmids Yeast unlike bacteria can support the propagation of more than one plasmid having the same replication origin i e there is no plasmid incompatibility issue in yeast Thus in a cotransformation experiment the efficiency of transforming each type of plasmid should remain at 10 per ug of DNA as determined by the number of colonies growing on SD medium that selects for only one of the plasmids The cotra
130. tion medium for the strains being assayed 4 Incubate the fresh culture at 30 C for 3 5 hr with shaking 230 250 rpm until the cells are in mid log phase of 1 ml 0 5 0 8 Record the exact OD when you harvest the cells Note Before checking the OD vortex the culture tube for 0 5 1 min to disperse cell clumps 5 Place 1 5 ml of culture into each of three 1 5 ml microcentrifuge tubes Centrifuge at 14 000 rpm 10 000 x g for 30 sec 6 Carefully remove supernatants Add 1 5 ml of Z buffer to each tube and vortex until cells are resuspended 7 Centrifuge cells again and remove supernatants Resuspend each pellet in 300 ul of Z Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 27 Yeast Protocols Handbook Vi a and p Galactosidase Assays continued buffer Thus the concentration factor is 1 5 0 3 5 fold Note Differences in cell recoveries after this wash step can be corrected for by re reading the OD of the resuspended cells 8 Transfer 0 1 ml of the cell suspension to a fresh microcentrifuge tube 9 Place tubes in liquid nitrogen until the cells are frozen 0 5 1 min 10 Place frozen tubes in a 37 C water bath for 0 5 1 min to thaw 11 Repeat the freeze thaw cycle Steps 9 amp 10 two more times to ensure that the cells have broken open 12 Set up a blank tube with 100 ul of Z buffer 13 Add 0 7 ml of Z buffer B mercaptoethanol to the reacti
131. to 40 ul of electrocompetent cells on ice Transfer samplesto a prechilledcuvette having a0 1 cm gap Performthe electroporation according to the manufacturer s instructions d Add 1 ml ofLB or preferably SOC medium vvith no antibiotic to the cuvette Transfer the cell suspension to a 14 ml conical Falcon tube Incubate at 37 C for 1 hr with vigorous shaking 250 rpm Pellet cells by centrifuging at 2 500 rpm for 5 min in a tabletop centrifuge Discard supernatant and resuspend pellet in residual liquid Plate cells on supplemented M9 amp agar medium Incubate plates at 37 C for 24 hr LB amp selection only or for 36 48 hr for nutritional selection on M9 medium If you do not recover any colonies see the Troubleshooting tips below i See Section C 3 for tips on plasmid isolation 2 Procedure for transforming chemically competent E coli KC8 Transformation efficiency is significantly affected by temperature Therefore prechill the 14 ml Falcon tubes and pipette tips to 4 C before using them a Prepare the chemically competent cells or thaw them on ice b Add 10 ul of yeast plasmid solution to a prechilled Falcon tube e o o c Add 100 ul of competent cells to the tube and mix well by gently tapping the tube d Incubate on ice for 30 min e Heat shock by transferring the tube to a 42 C water bath and incubating for 45 50 sec f Chill on ice for 2 min g Add 1 ml of LB broth or preferably SOC me
132. tomatic hot start when used with Taq or KlenTaq DNA Polymerase Kellogg etal 1994 Antibody mediated hot start withTaqStart Antibody is more convenient than manual hot start or wax bead mediated hot start and has been proven to be at least as effective as the conventional methods 5 Template quality a Because of the exponential nature of PCR amplification many conventional PCR applications such as screening cDNA inserts work well with templates of average or even low quality including plasmid DNA isolated from yeast Use 1 2 ul of yeast plasmid DNA preparation from Section VII B per PCR b Be sure to use a single well isolated yeast colony when inoculating liquid cultures for preparation of plasmid from yeast Chapter VII B c If the yeast transformant contains more than one plasmid insert sequence you may see multiple PCR bands Restreak the yeast transformant on the appropriate SD medium that maintains selection on the desired plasmid s but not on their interactions Appendix E The extra generations of growth will allow segregation i e loss of some of the plasmids After reconfirming the presence of positive plasmids using a B gal colony lift assay repeat the plasmid isolation and PCR analysis on well isolated colonies In some cases it may be necessary to transform E coli with the yeast plasmid prep and isolate plasmid from E coli transformants to ensure a homogeneous plasmid preparation Chapter VII C 6 Tips for
133. tore at 4 C for up to 2 months or at 20 C for up to 6 months If colloidal material precipitates mix the solution by inversion before using e Recommended CHROMA SPIN 1000 DEPC H O Columns Cat No 636093 and 2 ml centrifuge tubes for use with the columns e If you do not use CHROMA SPIN Columns you will need materials to perform phenol chloroform extraction and ethanol precipitation Phenol chloroform isoamyl alcohol 25 24 1 See Sambrook et al 1989 for information on preparing neutralized phenol solutions e 10M ammonium acetate 95 100 ethanol Clontech Laboratories Inc www clontech com Protocol No PT3024 1 34 Version No PR742227 Yeast Protocols Handbook VILVVorking With Yeast Plasmids continued 1 Prepare yeast cultures for lysis Step a b or c below a From a solid patch of growth i Spread a thin film of yeast cells 2 cm patch onto the appropriate SD agar medium ii Incubate plate at 30 C for 3 4 days The patch should show abundant yeast growth ili Scrape up a portion of the patch 10 mm and resuspend the cells in 50 ul of sterile H O or TE in a 1 5 ml microcentrifuge tube b From a liquid culture i Inoculate a large 2 4 mm fresh 2 4 day old yeast colony into 0 5 ml of the appropriate SD liquid medium Vortex tube vigorously to completely break up the colony and resuspend the cells ii Incubate at 30 C overnight with shaking at 230 250 rpm iii Spin down the cells by
134. trol the initiation of transcription of adjacent structural genes via binding of specific regulatory proteins An example is the binding of the yeast GAL4 transcriptional activator or DNA BD to the UAS of the GAL7 promoter wild type The genotype or phenotype of an organism as it is found in nature or in a standard laboratory strain Protocol No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 51 Yeast Protocols Handbook APPENDIX B Yeast Genetic Markers Used in the Matchmaker Systems TABLE IV SELECTED YEAST GENES AND THEIR ASSOCIATED PHENOTYPES Allele Wild type Mutant Phenotype of mutant TRP1 trp 1 901 Trp Requires tryptophan Trp in the medium to grow i e is a Trp auxotroph LEU2 leu2 3 112 Leu Requires leucine Leu to grow i e is a Leu auxotroph HIS3 his3 200 His Requires histidine His to grow i e is a His auxotroph URA3 ura3 52 Ura Requires uracil Ura to grow i e is a Ura auxotroph LYS2 lys2 801 Lys Requires lysine Lys to grow i e is a Lys auxotroph ADE2 ade2 101 Ade Requires adenine Ade to grow i e is an Ade auxotroph in addition confers a pink or red colony colorto colonies growing on media low in adenine The red pigment is apparently an oxidized polymerized derivative of 5 aminoimidazole ribotide which accumulates in vacuoles Smirnov etal 1967 Weisman et al 1987 GAL4 gal4 542 Gal Deficient in regulation of galactose metabolizing genes
135. ty of genes located on the same DNA molecule often via binding of regulatory proteins or factors confluent When yeast or bacterial colonies growing on an agar plate are so numerous that the edges of the colonies touch each other clone a A group of genetically identical cells or individuals derived by asexual division from a common ancestor b A heterologous cDNA fragment inserted into a vector also refers to copies of that original cDNA colony A visible clone of cells growing on solid medium diploid In yeast a cell having two complete chromosome sets as a result of mating of haploid a and a strains A cell can also be diploid for one particular gene or several genes due to the presence of plasmids or as a result of gene duplication dropout DO supplement A mixture of several amino acids and nucleosides that must be added to minimal synthetic medium to support the growth of yeast strains that have defined nutritional requirements typically one or more specific nutrients is left or dropped out of the DO supplement so that the resulting synthetic dropout SD medium will only support the growth of yeast that are able to synthesize that nutrient gene a The fundamental physical unit of heredity recognized through its variant alleles b a DNA sequence that regulates and encodes a functional product e g a polypeptide chain or an RNA molecule genetic complementation The production of a wild type phenotype when
136. uneven spreading of the cell suspension and will delay absorption of the liquid X gal is heat labile and will be destroyed if added to hot i e gt 55 C medium Prepare the required number of plates in advance Allow plates to dry unsleeved at room temperature for 2 3 days or at 30 C for 3 hr prior to spreading or streaking the cells Excess moisture on the agar surface can lead to uneven spreading of cells 1 Streak replica plate or spread the transformants to be assayed on selection medium containing X gal and BU salts When performing a two hybrid library screening where very few of the cotransformants are expected to be positive for lacZ expression or where it is difficult to predict the number of interactors plate the cells at a high density We recommend plating at two different densities to cover a range e g 0 5 x 105 cfu on some 150 mm plates and 2 x 10 on others e When performing a two hybrid assay where most or all of the individual colonies may be LacZ spread 200 400 cfu per 100 mm plate 2 Incubate plates at 30 C for 4 6 days 3 Check plates every 12 hr up to 96 hr for development of blue color Notes e If you are performing a two hybrid library screening using the Matchmaker LexA System please see the User Manual for further information on identifying and storing LacZ colonies Colonies grown on X gal containing medium will be somewhat smaller than those grown without X gal C Colo
137. urther information on library screening strategies and specific protocols Integration vs nonintegration of yeast plasmids For most yeast transformations performed while using the Matchmaker Systems it is not necessary or desirable to have the plasmid integrate into the yeast genome In fact yeast plasmids do not efficiently integrate if they carry a yeast origin of replication and are used uncut However there are two exceptions to this general rule as explained in the respective system specific User Manuals a In the Matchmaker One Hybrid System the researcher must Clontech Laboratories Inc www clontech com Protocol No PT3024 1 18 Version No PR742227 Yeast Protocols Handbook V Yeast Transformation Procedures continued construct their own custom reporter plasmid and then integrate it into the yeast host strain before performing the one hybrid assay b In the Matchmaker LexA Two Hybrid System the p8op lacZ reporter plasmid can be used either as an autonomously replicating plasmid or as an integrated plasmid depending on the desired level of reporter gene expression The primary reason for integrating a plasmid in some Matchmaker applications is to generate a stable yeast reporter strain in which only one copy of the reporter gene is present per cell and thereby control the level of background expression If you have an application that requires integration of a plasmid into the yeast genome please see Section V D Tran
138. ution for 1 ml assays and 10X Stop Solution for 200 ul assays 10 Record the optical density of each sample at 96 vvell plate 1 5 ml cuvette 410 nm OD a Add a corresponding volume of sterile unused culture media to the reagent blank 5 Zero the spectrophotometer using the reagent blank and measure the OD of the experimental and control samples relative to the blank 1 Calculate a galactosidase units One unit of a galactosidase is defined as the amount of enzyme that hydrolyzes 1 umole p nitrophenyl a p galactoside to p nitrophenol and D galactose in 1 min at 30 C in acetate buffer pH 4 5 Lazo et al 1978 a galactosidase milliunits ml x cell OD X V x 1 000 e x b x tx V x elapsed time in min of incubation a II V final volume of assay 200 ul or 992 ul V volume of culture medium supernatant added 16 ul or 8 ul OD optical density of overnight culture sxb p nitrophenol molar absorbtivity at 410 nm x the light path cm 10 5 ml umol for 200 ul format gt 16 9 ml u mol for 1 ml format where b 1 cm The optical density at 600 nm recorded in Step 2 is used to normalize the OD of different media samples to the number of cells in each culture The molar absorbtivity though independent of concentration and light path varies with the chemical properties e g pH of the solution Because different strengths and quantities of Stop Solution are used to terminat
139. vortex 2 X 1 min at room temperature placing the tube on ice for 30 sec in between the two vortexings c Transfer the liquid above the glass beads second cell extract to the corresponding first cell extract from Step 5 7 Allow any carryover glass beads to settle in the combined cell extracts 1 min then transfer the liquid above the glass beads to a fresh prechilled 1 5 ml screw cap tube 8 Pellet the proteins in a microcentrifuge at 14 000 rpm for 10 min at 4 C Carefully remove supernatant and discard 10 Quickly spin tubes to bring down remaining liquid Remove and discard liquid using a pipette tip 11 Resuspend each pellet in TCA Laemmli loading buffer Use 10 ul of loading buffer per ODs unit of cells Note If too much acid remains in the sample the bromophenol blue in the buffer will turn yellow Generally this will not affect the results of the electrophoresis 12 Place tubes in a 100 C boiling water bath for 10 min 13 Centrifuge samples at 14 000 rpm for 10 min at room temperature 20 222 14 Transfer supernatant to fresh 1 5 ml screw cap tube 15 Load the samples immediately on a gel Alternatively samples may be stored on dry Protocol ice or in a 70 C freezer until you are ready to run them on a gel No PT3024 1 www clontech com Clontech Laboratories Inc Version No PR742227 15 Yeast Protocols Handbook IV Preparation of Yeast Protein Extracts continued Cell pellets
140. y OOQpuEH Sio1989A LUA SAS pluqAH OAAL PIVD 4 1BeuuU9 8 A 8u1 u p piAoid ase s u nb s SOI s Buluol pue sdew uololi41 1 uolguuojul 40199A EUOILPPY q Ajayesedas ajqejiene osje aie spiluse d uuos L EO9L ON 182 uu91s S pluq H 9uO 1 JLWYNLN HL WIN 08089 ON 352 WaISAS PLIGAH OMLI JLWYNLN HZ VIVO 1 9091X ON 189 Waishs PLiqAH OM Jaye HZ P1V9 1 6091 ON 382 uu91s S pluqAH OAML VX81 1 LWYNPN HZ VX81 L 7O9L ON 189 Z WaIsAS pPIQ H OML19 JE WYNLN Z HZ PIV suonelreiqge WajsAs o Ady e 6 0 9 S due LdYL 2 2 V V 1661 1e 19 uolss u qns ul III pu H 49 daj aa vna z7y HZ e6pugd 202034 20 87 29 dwe ESIH 661 29 SN D 096681 III pu H 201 SIH ag vNa Vyx Hz vx r 9661 8 1 4 1 29 Z 0 dwe ESIH 9661 2 18 siuu oD uolssiuiqns u III pu H 9 9 SIH ag vNa 02 2 epid 6 0 97 dwe 1441 eg661 Je j ueg 9v9L0N III pu H 45 daj ag vna p7v9 Hz r1v5 61g5d L66L Je 19 181no1 6 0 71 6 b Be do id SA 9 suey LdYL 12 uolssiuuqns u IIIpu H EZ daj qg vNq z7y 6 2 FTV 114800 870 1 WH dwe 2 137 12 uolssiuuqns u IIIpu H 0 8 ney av p75 HZ V1Y O LLav5d 60 3 0 L 9 dwe 27747 6661 8 19 1s 9V UL

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