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1. a s 191luod tu e d41ou r ul s4S ulus UOHPULO SUBAL SIW31SAS GIY8AH OML QNV 3NO SHL NI SNIVYLS 31 4 LSV3 A XI 318V L page 61 TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 Technical Support Protocol PT3024 1 Version PR7X265 Advantage is a registered trademark of CLONTECH Laboratories Inc CHROMA SPIN Ligation Express QUICK Clone TaqStart YEASTMAKER and YEXpress are trademarks of CLONTECH Laboratories Inc 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 1997 CLONTECH Laboratories Inc All rights reserved
2. VII Working With Yeast Plasmids A General information Isolating plasmid DNA from yeast is nottrivial 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 our YEASTMAKER Yeast Plasmid Isolation Kit K1611 1 This procedure which was modified from the method of Ling et a 1995 uses extensive digestion with lyticase to weaken the cell walls and SDS to burst the resulting soheroplasts The DNA preps can be cleaned up using either CHROMA SPIN Columns or phenol chloroform extraction followed by ethanol precipita tion 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 samples simultaneously This purification method yields sufficient DNA of sufficient purity for use as a PCR template Chapter VIII or for transforming E
3. n n neq winipaw as uo uon l s 202 due ESIH 202 we 1aHL 48 4 Ypqy5 dwe 277 dY 188 892 7y O dwe zn371 av 188 89 p75 due eng ay 1884575 ue Z031 ay 888 JD dold vH dwe 13H1 erg Joyenijoe XlploE ZHAO dwe I dt 1 qg vNq Vz7y dolid vH gwe 2137 av 1888p V5 dolid vH 2031 ay 999 992 quohndu s q SHOL 3A NINOTO WALSAS G HSAH O AL H33VIWHOLVI IA 318V 1 1 6091 Vx91 1 6091 Hz vx q 1 GO9L HZ YTV s ue1q 1 NIN NIN 1 S09131 Hz YTV s ue q NIN 1 6091 Hz vx q 1 091 2 2 vWD s neq ININ 8 1 V091X Z HZ seueiqi NIN etu 9 s S vx 1d epi od 6Lapd 19 avod 4700 pevavod oravad avzrgd ol esvd 210 Lovd 10199 Protocol PT3024 1 TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 Technical Support Version PR7X265 page 58 CLONTECH Laboratories Inc Yeast Protocols Handbook tinued Ion con Plasmid Informati APPENDIX E BWS q 1no Jou ase YyoIyM nigeGd pue S HEGd woi way Ysinbuljsip o lqissod 1 soyew qolu OS BUS l uls e 20810 pue ISIHd UOH PPE l b 1 SIH GS UO e SE sn S slluu d
4. CLONTECH Laboratories Inc Yeast Protocols Handbook APPENDIX E nued i Plasmid Information cont oyx Aq no 104 51 WEgqyhHd lis LWS Buls e ALY YIM JO 1Z0e7d pue ISIHd Wo way sinSunsip o lqissod 1 seyew USIUA LWS Aq nd 104 ele nigesd pue SIHEGd q HO21NOTDO Je paylpow 1 A spiuse d peyeo pul ay u pequosep spiuuse d ay JO S AIIPAl D EYAd pue qd we yxeqd eg yxe7d 1 S NV d S NVTd L dverdd e 1661 7 Ja 670 ZL 2297 fiat dure Idul 1 091 6661 2 1uonqEM III PWH v6 d j 1 25 0 ul 065762 ecd uuunui Z HZ YTV L ewad 871 i dure Idul 1 S0913 661 72 1uonqEM III PWH v9 d j eLg9d u 106722 g d uunutu HZ VAd 9001 Zl r ee 8031 ZLOVd u 1909131 6661 90101 3 71 III PU H 001 n r 2778 u Bnue OVAS Z HZ vWD I q d 1661 7 4910 90 ZL EL 21 gwe 2131 4gavod u 1 S0913 6661 90191 3 1 III PUIH 0g1 n T 802778 u fnue e612 OVAS HZ Laid v LLHSd 0 E 0 9 dwe ESIH 1 60913 Y661 8 29 siuu9 joo III PU H 961 SIH u b uolsn Hz Vx 1 sod Vx 1d 20 670 ZS due ESIH VX Td ul L 6091 eg66L P jee III PWH 901 SIH 082 99 uwe ueuunH Hz VX91 uue1 Vx 1d z o z s S dwe ESIH 1 6091X 6661 TE 1uonqEM
5. III PWH Vil SIH vx q1d u 066 22 d uunu Hz VX91 gs vx 1d 70 980 60 Z 9 7 dwe dH 1 25 0 u v091 BE66 Je Ja jaueg III PWH 0 6 d l 087 990 uwe upuunH Z HZ vWD L SIWV1d 9 0 870 p dwe dil 61999 l 1 9091 88661 P l HEg III PUH 09 dij 087 999 ule ueuunH Hz SINV1d 9 2 dwe zn37 ay 8884 1 609 Ly 9661 7E on l HOOF 9 2 neq o pasny 8 eed uunuu HI WW ugsavod dwe zn31 1 091 81 92 711 AEAH P 0SdOA Ul 9 1 9091 6861 BuoS 9 splel4 691 n q ul u r in d p m 2 2 9 HZ 1661 8 19 400 970 970 60 771 EZ 976 due averad u 1 60913 6661 Spl l i 9 1 III PU 98 dij g0z 8 u Bnue 1 abel OVAS Hz Vx 1 1l avzvgd dwe eyn 1069 120610 ul s lis Sulpulq ggd L 09 Ly 9661 ye ON ql OUX HOOF v9 en JO 104102 4 pun 228 HI NN anqagesd due ey ESIH 0 1 9 siHd u seus ggd L 09 1b 9661 ye 1 ON ql OUX HOOF 29 SIH 22 JO 1041u00 ESIH HI NN SIH gd pe 8LHSd 6 1 12 9 dwe eyyn yxa 1 6091 9661 2 yelols III PU H 0l en 104102 228 Hz vx r zoe dogd 9u 1 4 1 u saus 4 H wnipew qS uondii5s q ui ls s e1O 9 9A s u l y zis uo uon l s SGINSV1d TOYULNOD ANY
6. J R amp Jones E W eds 1997 The Molecular and Cellular Biology of the Yeast Saccharomyces Cell Cycle and Cell Biology 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 promoter of Saccharomyces cerevisiae for efficient production of heterologous proteins Journal of Biotechnology 39 193 203 Ruohonen L Penttila M amp Keranen S 1991 Optimization of Bacillus o amylase Production by Saccharomyces cerevisiae Yeast 7 337 346 Sambrook J Fritsch E F amp Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Sadowski 1 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 Bio Techniques 20 960 962 Smirnov M
7. 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 coliby 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 H 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 Golemis 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 amp Schiestl R H 1992 Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res 20
8. 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 anew 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 specific transcriptional 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 and TATA regions can be switched to create novel promoters The mix and match nature of yeast TATA 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 acZ HIS3 and LEU2 reporter genes are under control of artificial promoter constructs comprised of a TATA and UAS or operator sequence derived from another gene Table l In some cases the TATA sequence and the UAS are derived from different genes indeed the LexA opera
9. prechilled 1 5 ml screw cap tube Pellet the proteins in a microcentrifuge at 14 000 rpm for 10 min at 4 C Carefully remove supernatant and discard Quickly spin tubes to bring down remaining liquid Remove and discard liquid using a pipette tip Resuspend each pellet in TCA Laemmli loading buffer Use 10 ul of loading buffer per ODsoo 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 Place tubes in a 100 C boiling water bath for 10 min Centrifuge samples at 14 000 rpm for 10 min at room temperature 20 222 Transfer supernatant to fresh 1 5 ml screw cap tube Load the samples immediately 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc IV Preparation of Yeast Protein Extracts continued E Troubleshooting Optimal electrophoretic separation of proteins depends largely on the quality ofthe 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 These same cons
10. until the cells are in mid log phase ODepo of 1 ml 0 5 0 8 Record the exact ODepo 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 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 ODsoo of the resuspended cells 8 Transfer 0 1 ml of the cell suspension to a fresh microcentrifuge tube Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued 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 thavv cycle Steps 9 amp 10 two more times to ensure that the cells have broken open 12 Set up a blank tube vvith 100 ul of Z buffer 13 Add0 7 mlofZ buffer B mercaptoethanol to the reaction and blank tubes Do not add Z buffer prior to fre
11. 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 Sons nc 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 8 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 amp Struhl K 1986 Functional dissection of a eukaryotic transcriptional protein GNN4 of yeast Cell 46 885 894 Ito H Fukada Y Murata K amp Kim
12. Binding Domain Sequencing Primer 6474 1 e GAL4 AD mAb 5398 1 e GAL4 DNA BD mAb 5399 1 e GAL4 DNA BD Vectors K1605 A K1604 B e GAL4 AD Vectors many LexA based two hybrid system and related products e MATCHMAKER LexA Two Hybrid System K1609 1 MATCHMAKER LexA Libraries many e LexA Monoclonal Antibody 5397 1 e MATCHMAKER pB42AD LD Insert Screening Amplimer Set 9108 1 e MATCHMAKER LexA DNA BD Insert Screening Amplimer Set 9109 1 pGilda LexA Vector 6183 1 General cloning reagents e QUICK Clone cDNA many Genomic DNA many E coli KC8 Electrocompetent Cells C2023 1 E coli KC8 Chemically Competent Cells C2004 1 e Ligation Express Kit K1049 1 Miscellaneous related reagents e X Gal 5 Bromo 4 chloro 3 indolyl 8 D galactopyranoside 8060 1 2 e Luminescent B gal Detection Kit II K2048 1 Advantage cDNA PCR Kit K1905 1 y e Advantage cDNA Polymerase Mix 8417 1 e CHROMA SPIN TE 400 Columns K1323 1 2 Protocol PT3024 1 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX A Glossary of Technical Terms Note Many ofthese 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 HIS3 is a wild type allele at the his3 locus In standard yeast nomenclature mutant alleles are written in lower case
13. DNA into the yeast genome n the LiAc transformation method yeast competent cells are prepared and suspended in a L Ac 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 fortransformants 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 intransformation efficiencies of 105 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 oftransforming each type of plasmid should remain at 105per ug of DNA as determined by the number of colonies growing on SD med
14. 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 therefore 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 acZ transcriptional signals that cannot be detected by X gal filter assays For a result it is more labor intensive than a filter assay However because of its greater sensitivity it is less likely to give a false negative result 1 2 Oo co page 30 Grow colonies on the appropriate SD selection medium 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 or reference plate of the colonies to be assayed Completely resuspend the colony in the Z buffer by repeatedly pipetting up and down Place tubes in liquid nitrogen for 0 5 1 min to freeze the cells Continue with Step 13 of the main procedure VI F above Compare results with those of the negative control Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc
15. allow diploid cells to form visible colonies 5 Score for grovvth 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 Section VI C Discard any B gal positive colonies that contain the candidate library plasmid alone Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 42 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc X References Yeast Two Hybrid System References Bartel P L 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 amp 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 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 Ac
16. are licensed from Baylor University CLONTECH encourages researchers not to redistribute the plasmids or yeast strains without prior written consent CG 1945 is the property of CLONTECH Laboratories Inc and should not be redistributed without prior written consent PJ69 2A is the property of the University of Wisconsin Research Foundation WARF and may not be redistributed without their prior written consent The Polymerase Chain Reaction PCR process is covered by patents owned by Hoffman LaRoche and F Hoffmann La Roche Ltd Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 3 Yeast Protocols Handbook CLONTECH Laboratories Inc I Introduction The Yeast Protocols Handbook provides background information and general yeast protocols that complement our system specific User Manuals The protocols in this Handbook have been optimized with our yeast based MATCHMAKER Two Hybrid and One Hybrid Systems and MATCHMAKER Libraries The Yeast Protocols Handbook is especially useful for researchers who wish to use yeast as avehicle fortheir 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 detailed information on c
17. 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 ll 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 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
18. cDNA 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 etal 1994 Ausubel etal 1994 In this procedure colonies are directly liftedonto a nylon membrane B glucuronidase is usedto break cell walls Reagents and Materials Required 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 HCl pH 7 5 6X SSC Ausubel et a 1994 2X SSC Ausubel et al 1994 100 000 units ml B glucuronidase type HP 2 crude solution from Helix pomatia Sigma 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 al 1994 1 If you have not done so already collect the colonies to be screened onto a master plate ina grid pattern to facilitate future
19. 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 coliand prepare plasmid using standard methods Sambrook etal 1989 Plasmid rescue via complementation of E co i 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 B Plasmid Isolation from Yeast Reagents and Materials Required The YEASTMAKER Yeast Plasmid Isolation Kit K1611 1 provides the SDS and lyticase solutions CHROMA SPIN 1000 DEPC H O 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 SDS Lyticase Solution 5 units ul in TE buffer store 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 Recommend
20. for yeast mating a b If you have not done so already generate an appropriate yeast strain containing the plasmid of interest 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 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 dropout to 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
21. 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 40 Dextrose glucose 40 Galactose for LexA Two Hybrid System D Galactose e g Sigma G 0750 40 Raffinose for LexA Two Hybrid System 1 mg ml 1000X CHX Cycloheximide Sigma C 7698 prepare in deionized H O and filter sterilize Store at 4 C for up to 2 months Store plates containing CHX sleeved at 4 C for up to 1 month e 50 mg ml kan kanamycin prepare in deionized and filter sterilize Store at 20 C indefinitely Store plates containing kan sleeved at 4 C for up to 1 month Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 51 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX C Media Recipes continued X gal 20 mg ml in DMF Dissolve 5 bromo 4 chloro 3 indolyl B D galactopyranoside X GAL 8060 1 in N N dimethylformamide Store in the dark at 20 C 10X Dropout DO supplements 10X dropout solutions contain all but one or more of the following components A combination of a minimal SD base and a DO supplement will produce a synthetic d
22. growth Prepare all reagents using sterile deionized distilled water such as Milli Q filtered Confirm that your water purification system is functioning properly Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays A General Information X gal must be used as the B galactosidase substrate for solid support assays because of its high degree of sensitivity X gal is 105 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 assay and all of the liquid 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 etal 1996 The colony lift filter assay Breeden amp Nasmyth 1985 is primarily used to screen large numbers of cotransformants that survive the H S3 growth selection in a GAL4 two hybrid or one hybrid library screeening t 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
23. 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 Length and G C content In general primers should have a T of at least 70 C to achieve 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 36 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VIII Analysis of Yeast Plasmid Inserts by PCR continued 3 Thermostable polymerase The Advantage 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 cDNA Polymerase Mix 4 Use of antibody mediated or conventional hot start To minimize nonspecific amplification we strongly recommend t
24. in liquid nitrogen for 0 5 1 min to freeze the cells Place frozen tubes in a 37 C water bath for 0 5 1 min to thaw Repeat freeze thaw cycle Steps 12 amp 13 once to ensure that cells have been cracked open Warm to room temperature enough reaction buffer for the entire experiment Set up a blank tube with 25 ul of Z buffer 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 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 Add 200 ul of Galacton Star reaction mixture to each sample tube or well and mix gently Incubate at room temperature 20 25 C for 60 min Note Light signals produced during this incubation are stable for gt 1 hr therefore detection can be performed 1 2 hr after the incubation 21 Centrifuge tubes at 14 000 rp
25. is a His auxotroph URA3 ura3 52 Uraz 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 color to colonies growing on media low in adenine The red pigment is appar ently an oxidized polymerized derivative of 5 aminoimidazole ribotide which accumulates in vacuoles Smirnov ef al 1967 Weisman et al 1987 GAL4 gal4 542 Gal Deficient in regulation of galactose metabolizing genes 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 CYHs2 2 Cyh Resistant to cycloheximide Reporter Gene Positive Negative Gene Description Phenotypea Phenotypea lacZ Encodes f galactosidase LacZ LacZ e Blue colony e White colony B gal activity above e Undetectable or background background level of B gal activity HIS3 Confers His prototrophy His His e Grows on 50 15 Does not grow on SD His LEU2 Confers Leu prototrophy Leu Leu e Grows on SD Leu Does not grow on SD Leu ADE2 Confers Ade prototrophy Ade Ade Grows on 50 Does not grow on SD Ade Pink or red colony color when grown on medium such as YPD lovv in Ade a Relative levels of background expression and reporter
26. 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 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 activity 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 ofthe 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 require ments typically one or more specific nutrients is left or
27. 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 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 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 to the tremendous 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 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 Alway
28. refer to your MATCHMAKER system specific User Manual for further information on library screening strate gies 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 Infact 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 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 18 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc V Yeast Transformation Procedures continued canbe 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 backg
29. spiuuse d s q u 401859 S H Ayee7 5 enueyy s s ul s4S puqAH UO HAYV NH LV N 94 l s u nb s SON ells Bujuojo pue sdew uoll9l11s 41 uolleuuojul 10199A EUOIIPPY q SUOHOE lUI l old VNQ l ds Huljosjep 104 115 lIod i se Asess u y o p sn uay 51 10nilsuo2 y spiuuse d 1 od y JO uo JO SON 24 olu p vol isnul u nb s yya 1 ql u nb s uoniuBoo u e l 1s S puq u uo ulul e dwe SYYN p r0 0 79 u ul l p uolo 1 20914 9661 IP On lQE EAB you I 04 HOOF v9 eIn JO 1041u02 4 pun Zoe HI WW 122210 due 0 1 u ul l p uolo 1 20914 8661 8 lq el ieA you 0 HOOF ys SIH JO 10409 s pun ESIH HI WW L ISIHd dwe SYYN p 0 1 9 u w j 1 2091 8661 Je lpUEX ly you OUX HOOF 19 SIH an JO 1041u09 4 puN ESIH HI WW ISIHd uoijss 55v as quondu s q wa shs e10 39A yuequ r s s 3 H ZIS uo uoh o l s onsoubeig SGINSVW1d H4140d44H NINOTO WALSAS 8 YSXVINHOLVW HA 318V1 page 59 TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 Technical Support Protocol PT3024 1 Version PR7X265
30. ul of a 200 mM stock solution Trypsin plasmin Sigma B6506 and thrombin Aprotinin 0 37 mg ml 120 ul of a 2 1 mg ml stock solution Some serine Sigma A6279 proteases Store the individual stock solutions as directed on the labels and follovv label precautions PMSF phenylmethyi sulfonyl fluoride stock solution 100X Dissolve 0 1742 g PMSF Sigma 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 VVear gloves Handle vvith care and read label precautions Glass Beads 425 600 um Sigma G 8772 For Urea SDS Protein Extraction Method page 54 Cracking buffer stock solution To prepare 100 ml Urea 8 M 48g SDS 5 wiv 5g Tris HCl pH6 8 40 mM 4 ml of 1 M stock solution EDTA 0 1 mM 20 ul of a 0 5 M stock solution Bromophenol blue 0 4 mg ml 40 mg 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 Crac
31. 0 Polyethylene glycol avg mol wt 3 350 Sigma P 3640 prepare with sterile deionized H gt O if necessary warm solution to 50 C to help the PEG go into solution 100 DMSO Dimethyl sulfoxide Sigma D 8779 10X TE buffer 0 1 M Tris HCl 10 mM EDTA pH 7 5 Autoclave 10X LiAc 1 M lithium acetate Sigma L 6883 Adjust to pH 7 5 with dilute acetic acid and autoclave C For B galactosidase Filter Assays Z buffer 7H O 16 1 g L NaHsPO m H20 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 B D galactopyranoside X GAL 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 B mercaptoethanol B ME Sigma M 6250 1 67 ml X gal stock solution D For Liquid galactosidase Assays with ONPG as Substrate 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 e ONPG o nitrophenyl B D galactopyranoside Sigma 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 Prepare solution fresh before each use Technical Support TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 Protocol PT3024 1 page 56 Vers
32. 091 S661 ye 19 yelolszi gean Ldn Esiy engl Ldn su Gen O V N Hz vx 1 Zoer 1y o 9Y gSIH V1V1eoIH SV U TVD ZSAT 0661 uolsuuof 9 yoli Zhao Z u o VOS E vpleb ZL 1 g gn 106 104 L 709 1 6661 P s dieH zn idi 2291 FSIH 108 CS I LO L Z PE 00Z ES Y Zg gEIn CLYW Z HZ vv 06LA Zoe VWL oag y vun SIH Y 117v9 SYL7V9 ZSAT uoleolunululo euosi d 0 16 Zu o 24 2 gs 08lPB ZtG tIeb 21 1 Zn 106 10 1 09 1 F661 TE zn Ldn 2291 ESIH 108 29 1 101 Z pE 00Z 8SIU Z gEIn VW Z HZ vWD 61 Zoe 1yyp 9Y Ty YYN s ueiqr Ld VOSIE jaw VElE ZLL g z n 106 1 dil 9 1 091 8661 P 1 dieH ena idi Zoe 101 2 OPE O0Z ESIY ZG ERIN OlV N Z HZ YTV 281A p O e0seu qip vun eSIH YL Tyo Y TY ZSAT uoleolunuluioo euosi d 0116 O Zu o 889 08 6 ZtG pleb 21 1 Zn 106 104 1 S0913 y661 TE s holl i ene idi 2291 ESIH 108 29 1 101 C PE 00Z ES Y ZS n CLYW Hz ZAMH Zoer 1yyp SY Ty 8 S O9IE ZrS rlEB upo ZLE Cn 106 1 d 1 S0913 661 TE s dieH ene ida Zoe 108 29 101 Z PE o0z Esly S Ein CLYW Hz 929 49 5998
33. 100 mm plates 125 mm filters e g VWR 28321 113 can be used with 150 mm plates Alternatively 85 and 135 mm filters can be specially ordered from Whatman Nitrocellulose filters also can be used but they are prone to crack when frozen Forceps for handling the filters Z buffer Appendix D Z buffer X gal solution Appendix X gal stock solution Appendix D Liquid nitrogen 1 2 3 4 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 B galactosidase assay below Use the SD selection medium appropriate for your system and plasmids When testing LexA transformants be sure to use gal raff induction medium Prepare Z buffer X gal solution as described in Appendix D For each plate of transformants to be assayed presoak a sterile Whatman 5 or VWR 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 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 106
34. 4 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 15 Yeast Protocols Handbook CLONTECH Laboratories Inc IV 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 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 lf 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 Ice cold 20 w v TCA in H O see Sambrook ef al 1989 for tips on preparing TCA solutions TCA Laemmli loading buffer Appendix D A Note Unless otherwise stated keep protein samples on ice 12 13 14 15 page 16 1 2 3 Thaw cell pellets on ice 10 20 min Resuspend each cell pellet in 100 ul of ice cold TCA buffer per 7 5 ODsoo units of cells For example for 33 total ODsoo units of cells use 0 44 ml of TCA buffer Place tubes on ice 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 col
35. 4 or 800 424 1350 page 25 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued 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 for the appearance of blue colonies Notes The time it takes colonies producing B galactosidase to turn blue varies typically from 30 min to 8 hr in a 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 reporter strains cotransformed with the positive controls for a two hybrid interaction give a positive blue signal within 60 min CG 1945 cotra
36. 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 BMC 884 308 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 11 Use the 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 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 ODgg of 1 ml 0 5 0 8 Record the exact ODepoo when you harvest the cells Note Before checking the OD vortex the culture tube for 0 5 1 min to disperse cell clumps Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 27 Yeast Protocols Handbook CLONTECH
37. 8 2412 Campbell K S Buder A Deuschle U 1995 Interaction of p56l ck 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 Q Russell M Birch D Raymond J amp Bloch W 1992 Prevention of pre PCR mispriming and primer dimerization improves low copy number amplifications Nucleic Acid Res 20 1717 1723 Chuang S E Chen A L amp Chao C C 1995 Growth of E coli at low temperature dramatically increases the transformation frequency by electroporation Nucleic Acids Res 23 1641 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 43 Yeast Protocols Handbook CLONTECH Laboratories Inc X References continued D aquila 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 Touchdovvn
38. C or colder Figure 2 Urea SDS protein extraction method Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 13 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 1 5 ml screw cap microcentrifuge tubes Glass beads 425 600 um Sigma G 8772 Protease inhibitor solution Appendix D A PMSF stock solution Appendix D A Cracking buffer stock solution Appendix D A e Cracking buffer complete Appendix D A Note Unless otherwise stated keep protein samples on ice page 14 1 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 ODepo units of cells For example for 33 total ODsoo 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 e Because the initial excess PMSF in the cracking buffer de
39. CLONTECH Yeast Protocols Handbook PT3024 1 Protocol Supplement for Catalog Product K1605 1 MATCHMAKER Two Hybrid System K1604 1 MATCHMAKER Two Hybrid System 2 K1603 1 MATCHMAKER One Hybrid System many MATCHMAKER Libraries NL4000AA MATCHMAKER Random Peptide Library K1609 1 MATCHMAKER LexA Two Hybrid System many MATCHMAKER LexA Libraries 5398 1 GAL4 AD Monoclonal Antibody 5399 1 GAL4 DNA BD Monoclonal Antibody Please refer to the User Manual for system specific information and procedures PR7X265 FOR RESEARCH USE ONLY Yeast Protocols Handbook CLONTECH Laboratories Inc Table of Contents VI VII VIII X XI Introduction Introduction to Yeast Promoters Culturing and Handling Yeast Preparation of Yeast Protein Extracts General Information Preparation of Yeast Cultures for Protein Extraction Preparation of Protein Extracts Urea SDS Method Preparation of Protein Extracts TCA Method Troubleshooting m ea 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 11 Ow Galactosidase 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 Usi
40. D minimal medium to keep selective pressure on extrachromosomal plasmid s e The growth in YPD of yeast strains carrying the ade2 101 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 50 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 ODepoo 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 into fresh medium to produce an 6 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 ODsoo 0 4 0 6 Note Generally YPD 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 GAL1 promoter of a LexA system plasmid e g pB42AD or pGilda YPD contains glucose which represses trans
41. G GAL2 bs4 CGGGGCGGATCACTCCG GAL2 bsb CGGATCACTCCGAACCG UASg 17 mer CGGAAGACTCTCCTCCG Figure 1 Sequence of the GAL4 DNA BD recognition sites in the GAL1 and GAL2 UASs and the UASg 17 mer consensus sequence Giniger 8 Ptashne 1988 Technical Support TEL 650 424 8222 or 800 662 2566 CL0N Protocol PT3024 1 page 6 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc ll Introduction to Yeast Promoters continued 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 In the 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 contain one or more conserved palindromic sequences to which the GAL4 protein binds Guthrie amp Fink 1991 Giniger ef a 1985 reviewed in Heslot amp Gaillardin 1992 The 17 mer consensus sequence referred to here as UASg 17 mer functions in an
42. Handbook or the CLONTECH Catalog Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 4 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc II 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 GAL1 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 Molecular Biology by Guthrie amp Fink 1991 V2010 1 Molecular Biology and Genetic Engineering of Yeasts edited by Heslot amp Gaillardin 1992 Stargell amp Struhl 1996 and Pringle et al 1997 V2365 1 UAS and TATA regions are basic building
43. Laboratories Inc VI B Galactosidase Assays continued 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 7 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 ODepo 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 and the buffer blank to stop color development Record the stop time For very strong B galactosidase positive colon
44. MAKER LexA DNA BD Insert Screening Amplimers 9109 1 are for conventional PCR amplification of inserts in pLexA and pGilda B Tips For Successful PCR of Yeast Plasmid Templates 1 Optimization of thermal cycling parameters The optimal eycling parameters will vary with different templates primers experimental protocols tubes and thermal cyclers Refer to the LD Insert Screening Amplimers User Manual Ausubel etal 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 etal 1991 Roux 1995 Briefly touchdown PCR involves using an annealing extension temperature that is several degrees typically 3 10 C higherthan the Ta ofthe primers during the initial PCR cycles typically 5 10 The annealing extension temperature is then reduced to the primer T 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
45. N Smirnov V N Budowsky E 1 Inge Vechtomov S G amp Serebrjakov N G 1967 Red pigment of adenine deficient yeast Saccharomyces 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 Tornow J amp Santangelo G G 1990 Efficient expression of Saccharomyces cerevisiae glycolytic gene 1 is dependent upon a cis acting regulatory element UAS ppg 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 UASg Molecular and Cellular Biology 4 2467 2478 Yoccum R R 1987 The GAL1 Zand 10 upstream activator sequences are not enhancers in Biological Research on Industrial Yeasts Vol 3 Stewart G Russell 1 Klein R amp Hiebsch R Ed CRC Press Boca Raton Fl page 61 Protocol PT3024 1 Technical Support
46. NA repair proteins XPA and ERCC1 Proc Natl Acad Sci USA 91 5012 5016 Ling M Merante F amp Robinson B H 1995 A rapid and reliable DNA preparation method for screening alarge number of yeast clones by polymerase chain reaction Nucleic Acids Res 23 4924 4925 Technical Support TEL 650 424 8222 or 800 662 Fr a Protocol PT3024 1 page 44 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc X References continued Liu J Wilson 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 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 amp 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 amp 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 Pringle J R Roach
47. 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 colony used for the inoculum will result in slow growth see Section III 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 to YPD 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 10 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
48. TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 45 Yeast Protocols Handbook CLONTECH Laboratories Inc XI MATCHMAKER and Related Products page 46 Technical Support TEL 650 424 8222 or 800 662 2566 CL0N FAX 650 424 1064 or 800 424 1350 Product Cat General reagents for work with yeast e YEASTMAKER Yeast Transformation Kit K1606 1 e YEASTMAKER Carrier DNA K1606 A e YEASTMAKER Yeast Plasmid Isolation Kit K1611 1 YPD Medium 8600 1 YPD Agar Medium 8601 1 e Minimal SD Base contains glucose 8602 1 e Minimal SD Agar Base contains glucose 8603 1 e Minimal SD Base Gal Raf contains galactose amp raffinose 8611 1 e Minimal SD Agar Base Gal Raf contains galactose amp raffinose 8612 1 Dropout DO Supplements for use with any SD Base many e YEXpress Yeast Inducible Expression Systems many e YEXpress Secretion Yeast Expression System 6200 1 GAL4 based one and two hybrid systems and related products e Mammalian MATCHMAKER Two Hybrid Assay Kit K1602 1 e MATCHMAKER One Hybrid System K1603 1 e MATCHMAKER Two Hybrid System K1605 1 e MATCHMAKER Two Hybrid System 2 K1604 1 e Two Hybrid cDNA Library Construction Kit K1607 1 MATCHMAKER cDNA Libraries many MATCHMAKER Pretransformed Libraries many e MATCHMAKER DNA BD Insert Screening Amplimer Set 5417 1 MATCHMAKER AD LD Insert Screening Amplimer Set 9103 1 e GAL4 Activation Domain Sequencing Primer 6473 1 e GAL4
49. Their Associated Phenotypes 49 Table V MATCHMAKER Reporter Genes and Their Phenotypes 49 Table VI MATCHMAKER Two Hybrid System Cloning Vectors 58 Table VII MATCHMAKER One Hybrid System Cloning Reporter Plasmids 59 Table VIII MATCHMAKER Reporter and Control Plasmids 60 Table IX Yeast Reporter Strains in the MATCHMAKER One and Two Hybrid Systems 61 List of Figures Figure 1 Sequence of GAL4 DNA BD recognition sites in the GAL1 and GAL2 6 UASs and the UASg 17 mer Figure 2 Urea SDS protein extraction method 13 Figure 3 TCA protein extraction method 15 Notice to Purchaser Practice of the two hybrid system is covered by US Patents 5 283 173 and 5 468 614 assigned to the Research Foundation of the State University of New York 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 New York 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 Carol Dempster Ph D at the Long Island Research Institute for license information Tel 516 361 6800 Fax 516 361 6840 All plasmids except for pACT2 and pAS2 1 are licensed from The Research Foundation of the State University of New York pACT2 pAS2 1 and yeast strains Y187 and Y190
50. Yeast Transformation Procedures continued F Troubleshooting Yeast Transformation The overall transformation efficiency should be at least 104 cfu ug for transformation with a single type of plasmid and 102 cfu ug for simultaneous cotransformation with two types of plasmids If your cotransformation efficiency is lower than expected calculate the transformation efficiency of the single plasmids from the number of transformants growing on the 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 page 22 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 use YEASTMAKER Carrier DNA which is available separately K1606 A or as part of the YEASTMAKER Yeast Transformation System K1606 1 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
51. ad 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 Sons Inc Chapters 20 0 and 20 1 Golemis E A Gyuris J amp Brent R 1994 Interaction trap tvvo 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 a human 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
52. 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 GAL and GAL2 UASs and the UAS 17 mer 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 Inthe GAL4 based MATCHMAKER Two Hybrid Systems either an intact GAL UAS which contains four GAL4 binding sites 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 1 The H S3 reporter of PU69 2A HF7c and CG 1945 and the JacZreporter of Y190 Y187 and SFY526 are all tightly regulated by the intact GAL7 promoter including the GAL7 and GAL 1 minimal promoter In HF7c and CG1945 lacZ expressi
53. ambrook etal 1989 Appendix E 5 Even if you have a substantial amount of DNA in your prep there is a remote possibility that the plasmid of interest 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 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 35 Yeast Protocols Handbook CLONTECH Laboratories Inc VIII Analysis of Yeast Plasmid Inserts by PCR A General information Sometimes a tvvo 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 amplifi
54. appropriate SD medium to select for colonies with an integrated reporter gene E Small scale LiAc Yeast Transformation Procedure 1 OQ W Inoculate 1 ml of YPD or SD 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 soo gt 1 5 Transfer 30 ml of overnight culture to a flask containing 300 ml of YPD Check the ODepo of the diluted culture and if necessary add more of the overnight culture to bring the ODsoo up to 0 2 0 3 Incubate at 30 C for 3 hr with shaking 230 rpm At this point the ODsoo should be 0 4 0 6 Note If the ODsoo is lt 0 4 something is wrong with the culture see Troubleshooting Section F 6 7 Place cells in 50 ml tubes and centrifuge at 1 000 x g for 5 min at room temperature page 20 20 21 C Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc V Yeast Transformation Procedures continued 8 Discard the supernatant and add 25 50 ml of sterile TE or distilled H2O to
55. ark at 4 C for up to two months Adjust final volume to 1L if necessary Notes e Galactose must be highly purified and contain lt 0 01 glucose 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 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 lf you are assaying for expression of a lacZ reporter gene in a system that requires expression of a protein from an intact yeast GAL1 promoter such as in the MATCHMAKER LexA Two Hybrid System you must use 2 galactose 1 raffinose 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 L Tryptophan 200 mg L T 0254 L Tyrosine 300 mg L T 3754 L Uracil 200 mg L U 0750 Stock solutions for use with SD Media 1M 3 AT 3 amino 1 2 4 triazole Sigma 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 10X BU Salts Dissolve the following components in 1 L total of HO 70
56. arry the CYH52 gene Therefore one can effectively select for yeast cells that have spontaneously lost the CYH52 bearing plasmid while retaining the other plasmid simply by plating the cotransformants on the appropiate SD medium containing cycloheximide Note The CYH2 gene encodes 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 40 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc IX Additional Useful Protocols continued 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 Cyh cotransformants of interest pick a colony 1 3 mmin 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 cyclohexim
57. ation using AD vector specific insert screening primers Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 33 Yeast Protocols Handbook CLONTECH Laboratories Inc VII Working With Yeast Plasmids continued 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 For transformation of electrocompetent cells you need an electroporator and a cuvette with a 0 1 cm gap Yeast plasmid DNA from Section B above Sterile 14 ml polypropylene conical tubes e g Falcon 2059 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 Materials for isolating plasmid DNA from E coli page 34 1 Procedure for transforming electrocompetent E coli KC8 a b c o o Prepare o
58. ays described here use one of three substrates ONPG CPRG or a chemilumi nescent subsirate Galacton Star The three substrates differ in their relative cost sensitivity and reproducibility See Table Ill To reduce variability in liquid B galactosidase assays assay five separate transformant colonies and perform each assay in triplicate It is important that the colonies to be assayed for B galactosidase activity are growing 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 lacz 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 lacZ reporter under the control of the inducible GAL1 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 23 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued TABLE III COMPARISON OF B GALACTOSIDASE ASSAYS Protocol Typeofassay Substrate Section Applications Comments In vivo X gal in medium VI B Less sensitive than colony liftassays recommended agar plat
59. best results we recommend using the Luminescent B galactosidase Detection Kit II K2048 1 which includes a reaction buffer containing the Galacton Star substrate and the Sapphire IITM accelerator positive control bacterial B galactosidase and a complete User Manual page 28 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued Chemiluminescent detection of B galactosidase t is important to 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 If in doubt determine the linear range of the assay and if necessary adjust the amount of 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 10 11 12 13 14 15 16 17 18 19 20 Prepare 5 ml overnight cultures in liquid SD medium as described in Chapter III A 3 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 se
60. cZ LexA Opis LexA GAL 1 high pHISi HIS3 none n a HIS3 TC TR n a leaky pHISi 1 HIS3 none n a HIS3 TC TR n a leaky pLacZi lacZ none n a HIS3 TC TR n a tight a See Appendices E amp 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 al 1993 Harper et al 1993 This is the minimal TATA region of the GAL1 promoter it does not include the GAL7 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 bs2 GAL1 bs3 GACTCTCCTCCGTGCGT GAL1 bs4 CGCACTGCTCCGAACAA GAL1 UAS GAL2 bs1 CGGAAAGCTTCCTTCCG GAL2 bs2 CGGCGGTCTTTCGTCCG GAL UAS GAL2 bs3 CGGAGATATCTGCGCC
61. cation 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 cDNA Polymerase Mix 8417 1 The Advantage cDNA PCR Kit K1905 1 provides a KlenTaq DNA Polymerase Mix which includes TaqStart M Antibody a 10X KlenTaq PCR reaction buffer dNTPs 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 lnsert 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 lnsert Screening Amplimers 9103 1 are for amplifying inserts in the GALA AD cloning vectors pGAD10 pGAD424 pGAD GL pGAD GH pACT and pACT2 MATCHMAKER pB42AD LD Insert Screening Amplimers 9108 1 are for amplifying inserts in the LexA system AD cloning vector pB42AD e MATCHMAKER DNA BD Vector Insert Screening Amplimers 5417 1 are for conventional PCR amplification of inserts in the GAL4 DNA BD cloning vectors pGBT9 pAS2 and pAS2 1 e MATCH
62. cription from the GAL1 promoter Technical Support TEL 650 424 8222 or 800 662 corer Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 whether 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 furthe
63. ction On the day of the experiment prepare the Galacton Star reaction mixture Keep buffer on ice until you are ready to use it Vortex the overnight culture tube for 0 5 1 min to 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 Incubate the fresh culture at 30 C for 3 5 hr with shaking 230 250 rpm until the cells are in mid log phase ODepo of 1 ml 0 4 0 6 Vigorously vortex the culture tube for 0 5 1 min to disperse cell clumps Record the 1 when you harvest the cells 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 Carefully remove supernatants Add 1 5 ml of Z buffer to each tube and thoroughly resuspend the pellet Centrifuge at 14 000 rpm 10 000 x g for 30 sec Remove the supernatants Resuspend each pellet in 300 ul of Z buffer Thus the concentra tion factor is 1 5 0 3 5 fold Read the ODggy of the resuspended cells The ODsoo 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 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 Place tubes
64. d 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 Stock solution may need to be warmed to 60 C to reliquefy 400 ul Recipe above 50 ul 20 ul PMSF stock solution 100X 20 ul Prechilled recipe above 30 ul TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 page 55 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 K1606 A 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 To prepare 10 ml of solution Final Conc PEG 4000 40 TE buffer 1X LiAc 1X Stock solutions 8 ml of 50 PEG 1 ml of 10X TE 1 ml of 10X LiAc 50 PEG 335
65. d 2096 TCA per 7 5 ODepo units of cells Note The volume of the glass beads can be measured using a graduated 1 5 ml microcentrifuge tube 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 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 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 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 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
66. d with pGilda and grown in the presence of glucose or galactose respectively April 1997 CLONTECHniques no data available for pB42AD Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 9 Yeast Protocols Handbook CLONTECH Laboratories Inc 11 Culturing and Handling Yeast For additional information on yeast vve recommend Guthrie and Fink 1991 Guide to Yeast Genetics and Molecular Biology V2010 1 A Yeast Strain Maintenance Recovery from Frozen Stocks and Routine Culturing 1 Long term storage e Yeast strains can be stored indefinitely in YPD 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 Appendix E for plas page 10 mid 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 w
67. 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 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 in the promoter region In yeast
68. e only when the cells to be assayed contain many copies of the lacZ reporter gene such as on a high copy number plasmid e 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 e Relatively sensitive recommended when the cells filter to be assayed contain one or only a few copies of the lacZ reporter gene e Convenient for large scale experiments screen many plates and colonies at the same time 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 asmall number of selectedtransformants 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 VLE e Forassaying asmallnumber ofselectedtransformants 10 times more sensitive than ONPG e Potential drawbacks Less reproducible than ONPG for strong positive colonies because of CPRG s fast reaction rate Liquid culture Chemiluminescent VI F e Forassaying asmall number of selectedtransforman
69. e 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 ef al 1989 To prepare agar plates 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 4 ml of a 10 mg ml stock of proline e Stock solutions for use with M9 or LB media Ampicillin 50 mg ml in HO 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 PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 53 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX D Solution Formulations A ForPreparation of Protein Extracts 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 P4265 in DMSO Leupeptin 0 03 mM 2 ul of a 10 5 mM stock solution Some thiol and Sigma L2884 serine proteases Benzamidine 145 mM 500
70. ed CHROMA SPIN 1000 DEPC H O Columns K1334 1 and 2 ml centrifuge tubes for use with the columns 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 10 M ammonium acetate 95 100 ethanol Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 31 Yeast Protocols Handbook CLONTECH Laboratories Inc VII Working 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 iii 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 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 upthe colony and resuspend the cells ii Incubate at 30 C overnight with shaking at 230 250 rpm iii Spin down the cells by centrifuging at 14 000 rpm for 5 min iv Carefully pour off the supernatant and res
71. efined minimal medium lacking one or more specific nutrients The specific components omitted depends on the selection medium desired To prepare SD Trp Leu for example use a 10X dropout supplement lacking Trp and Leu If a component is not indicated as missing then it is assumed to be present in the medium Many of the commonly used 10X dropout supplements can be purchased from CLONTECH If you prefer you can prepare your own DO supplements as described below 10X dropout supplements may be autoclaved and stored at 4 C for up to 1 year Note Serine aspartic acid and glutamic acid have been left out of this recipe because they make the media too acidic The yeast can synthesize these amino acids endogenously 10X Concentration Sigma Cat L lsoleucine 300 mg L l 7383 L Valine 1500 mg L V 0500 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 9511 L Leucine 1000 mg L L 1512 L Lysine HCI 300 mg L L 1262 L Methionine 200 mg L M 9625 L Phenylalanine 500 mg L P 5030 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 52 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX C Media Recipes continued B E coli MEDIA Hanahan s SOC Medium Final concen
72. er 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 39 Yeast Protocols Handbook CLONTECH Laboratories Inc IX Additional Useful Protocols continued 10 Carefully layer the nylon membrane colony side up on top of the B glucuronidase soaked filter Avoid trapping air bubbles 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 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 gt 60X 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 6X 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
73. erile glass beads 5 7 beads per 100 mm plate to promote even spreading of the cells D Integrating Plasmids into the Yeast Genome Important Please read Section V A for guidelines 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 e Ifthe 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 The vector 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
74. es 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 e Because of its 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 Leu 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 in the medium to reduce background growth Any of the common E coli host 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 colitransformant 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 PCR amplific
75. esearchers 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 K1606 1 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 antibodies and sequencing primers and yeast media including Minimal SD Base and many different formulations of Dropout DO Supplement Our YEXpress Yeast Expression Systems are ideal for expressing and characterizing positive clones identified in a one or two hybrid library screening For example pYEX BX 6199 1 has the copper inducible CUP1 promoter driving expression of cloned proteins pYEX 4T 1 2 and 3 46196 1 6197 1 and 6198 1 respectively have CUP1 promoters driving expression of GST fusion proteins pYEX S1 6200 1 is used for expression and secretion of cloned proteins including GST tagged proteins For ordering information on these products please see Chapter XI of this
76. et 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 program Consult your scintillation counter s manufacturer for further information about this software For detection methods described in Steps 22 24 Calculate the B galactosidase activity in terms of RLU ODggq unit of cell culture Note that Miller unit calculations are not possible using these methods 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 in the unknown samples using the standard curve Determine the amount of enzyme per OD oo unit of cell culture The final ODsoo units of cells assayed per sample is calculated as follows ODepoo from Step 5 x vol from Step 18 x conc factor from Step 9 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 well 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 senstive than a luminometer 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
77. evel expression due to the GAL1 UAS when induced by a positive two hybrid interaction this construct also exhibits a significant level of constitutive leaky expression due to the H S3 TC In contrast in HF7c and CG 1945 the entire H S3 promoter including both TATA boxes was replaced by the entire GAL1 promoter leading to tight regulation of the H S3 reporter in those strains Feilotter ef al 1994 The H S3 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 HIS3 TC The leaky H S3 expression of these one hybrid plasmids is first Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 7 Yeast Protocols Handbook CLONTECH Laboratories Inc ll Introduction to Yeast Promoters continued used to help construct H S3 reporter 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 integra
78. ezing 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 e The time needed will vary 83 15 min for the single plasmid gal positive control 30 min for a two hybrid positive control and up to 24 hr for weaker interactions 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 at Ayo and measure the of the 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 o t x V x ODeoo where t elapsed time in min of incubation V ml x concentration factor ODgo0 Agoo Of 1 ml of culture The concentration factor from Step D 7 is
79. f athermostable DNA polymerase during repeated cycles of heating and cooling phenotype The observable properties of an organism determined by the organism s genetic constitu tion 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 which 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 own Trp from other biomolecules and do not require it in the medium A prototrophic transformation 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 tran
80. f the appropriate sterile 10X dropout solution or the required amount of DO Supplement powder see package instructions e Adjust 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 Add the appropriate sterile carbon source usually dextrose glucose to 2 unless specified otherwise for your application Adjust final volume to 1L if necessary Notes e 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 that SD Minimal Base from CLONTECH already contains a carbon source If you purchase galactose separately it must be highly purified and contain lt 0 01 glucose 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 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 e
81. 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 the mixed plasmid DNA from yeast use it to transform E coli isolate the desired plasmid from E coli transformants and transform the desired yeast host strain with the isolated plasmid DNA Alternatively the yeast cotransformant strain can be grown for several generations on SD medium that maintains selection on the 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 K1604 1 but cannot be used with the host strains provided with Pretransformed MATCHMAKER Libraries or the original MATCHMAKER System K1605 1 1 Segregation by natural loss of an unselected plasmid a Culture individual cotransformant colonies Separately in 3 ml of the appropriate SD liquid selection medium for 1 2 da
82. forming E coli with 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 Hovvever if you choose to use chemically competent cells Section C 2 itis essential that the cells be able to yield a transformation efficiency of at least 107 cfu ug of pUC19 DNA Nutritional selection of E coli transformants n the MATCHMAKER two hybrid systems cloning vectors carrying H S3 LEU2 or TRP1 markers can be selectively rescued by complementation of the E coli hisB leuB or troC mutations respectively The yeast H S3 LEU2 and TRP1 genes are expressed well enoughin E colito allow this complementation Furthermore due to incompatibility of the E coliplasmid 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 C2004 1 and Electrocompetent C2023 1 Cells are available from CLONTECH HB101 which carri
83. gene induction are dependent on the promoter constructs controlling them See Chapter II for information on the promoters b 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 49 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX C Media Recipes A YEAST MEDIA e YPD medium YPD Medium 8600 1 and YPD Agar Medium 8601 1 are available in convenient 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 f you purchase CLONTECH s YPD media prepare the medium according to the instructions provided If you prefer you can prepare your own YPD mixture as follows 20 g L Difco peptone 10 g L Yeast extract 20 g L Agar for plates only Add H O to 950 ml Adjust pH to 5 8 if necessary and autoclave Allow medium to cool to 55 C and then add dextrose glucose to 2 50 ml of a sterile 40 stock solution 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 performa
84. grades 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 ODepoo 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 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 Transfer the supernatants to fresh 1 5 ml screw cap tubes and place on ice first 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 crack
85. hat you perform hot start PCR There are several methods available for hot start PCR including those using wax beads Chou et al 1992 or a manual hot start D aquila ef al 1991 TaqStart Antibody 5400 1 2 provides an automatic hot start when used with Taq or KlenTaq DNA Polymerase Kellogg et al 1994 Antibody mediated hot start with TaqStart 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 applica tions 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 f 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
86. ide selection to work 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 host strain For example use 1 0 ug ml for CG 1945 10 0 ug ml for Y190 Incubate the plate at 30 C until individual Cyh colonies appear This usually takes 3 5 days Transfer the Cyh colonies to appropriate SD selection plates to verify that they have lost the CYH52 bearing plasmid and retained the plasmid of 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 ef al 1993 Finley amp Brent 1994 See Guthrie amp Fink 1991 or Pringle et al 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
87. 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 of colonies to be screened presoak a Whatman 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 B 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 filt
88. iderations 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 ef al 1987 96 1 Few or no immunostained protein bands on the blot Thetransfer of protein bands to the blot may be confirmed by staining the blot with Ponceau S The presence of protein bands in the gel before transfer may be confirmed by staining aparallellane 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 Theextentofcellwall 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 thatin Steps C 8 a and D 12 boiling the protein extracts the samples are placed into a water bath that is already boi
89. ies color development occurs within seconds weak to moder ate 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 ODs7 of the samples An ODs7 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 ODz z t x V x ODeoo where t elapsed time in min of incubation V 0 1 x concentration factor 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 II Liquid nitrogen Luminometer or scintillation counter with single photon counting program Optional 96 well opaque white flat bottom microtiter plates WBPO05 Optional Purified B galactosidase for a standard curve Note For
90. ify 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 doing so use electrotransformation rather than chemical transformation higher transformation efficiencies are usually obtained with electroporation 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 If you 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 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 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 S
91. ing 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc IV Preparation of Yeast Protein Extracts continued Cell pellets 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 First Cell Extract liquid above beads e Place on ice Beads and unbroken cells e Add ice cold 20 TCA e Bead beat cells 1 x 30 sec or vortex for 5 min at 4 C e Combine Cell Extracts e Allow glass beads to settle 1 min Beads Second Cell Extract and unbroken cells liquid above beads discard Beads and unbroken cells discard Combined Cell Extracts liquid above beads e Centrifuge at 14 000 rpm for 10 min Pellet 5 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 Immediately load gel or Pellet discard Protein extract freeze at 70 C or colder Figure 3 TCA protein extraction method Protocol PT302
92. ion PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 9 g L Aspartate hemi Mg salt Sigma A 9506 0 065 g BSA 1 0g 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 Store at 4 C for up to 3 months e 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 57 CLONTECH Laboratories Inc Yeast Protocols Handbook APPENDIX E Plasmid Information liS IPS 8 uleluo2 Jou s op OLQV d 25 uons Bip Aq p zue ul si pzvaVvOd p 1 ZSVvd ul nbiun si ays H097 94L 1 12 sem y 99u81J8J91 S y u p quos p plluse d v AEAL P 8 SI zSVd SOUBIQIT H3MVINHO VN p uuoJsue1liqd pue ulu 61 JOJOBA pue jenuelN JOSP l s4S PLIQAH OML VX 7 y 100QpUEH s1Jo 989A WEISS PLIQAH OML PTVD HAYV NH LV N 24 l s ocu nb s SON s Buluojo ajdiy
93. ips 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 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 ODsoo 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 st
94. ium that selects for only one of the plasmids The cotransformation efficiency is determined by the number of colonies growing on SD medium that selects for both plasmids and should be 104 cfu ug DNA Simultaneous cotransformation is generally preferred because it is simpler than sequential transformation and because of the risk that 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 in the 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 up to 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
95. jnw pue sdew uoll9l s 1 uolleuuojul 10199A PUOIIIDDV q H resed s qelieA e osje ase spiusejd awos 1 609 19 WeIshsS PLQAH 8UC HAMVWHOLVW HI WW 1 S09 EM uu9 s S H3MVIWHO1VN HZ YTV 1 60913 uu9ls S puq H oAL VX91 HAYV NHOLV N HZ VxX81 1 09 142 Z W JS S PUQAH OML HAMV NHOLV N Z HZ suoleineiqqe walsfs 0 Z N e 02020 0661 ye sun o 9661 TE 0490112 9661 ye 19 811 0 eg664 TE 19 6661 TE 19 1SI1 V ULA 661 72 9 1SI V ULA E 661 TE Ja j peg 6661 TE 42 aeg s yerd C661 ye sun o 6661 72 s dieH wwo suad oppaja V661 7249 wwo suad apala 6661 7 79 ul ulpu p u seid s u s aiqeyrene jou 979201 qe ieA jou qe ieA jou 217910 88LELN aiqeyrene jou Z6v0EN 66862 qe ieA jou uoiss 55v yuequey 20 87 S III PU H 9 Z 0 III PU H 6 0 III PU H 0 70 419 III PU H 60 910 1 2 III PU H p Z 0 6 G III PU H p Z 0 6 S III PU H Seo 970 42 976 III 270 6 0 9 III 8 0 67 III PUIH 9 1 SO S 0 6 I Hooz q s s zu onsoubeig 01 99 959 69 6 99 99 sy 9 v8 Vs 991 qv zis SIH SIH dil n 7 neq neq neq dij dil
96. king 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX D Solution Formulations continued For TCA Protein Extraction Method e 20 w v TCA in H O Store at 4 C See Sambrook et al 1989 for tips on preparing TCA solutions 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 HO 9 7 ml Protease inhibitor solution 50 ul ml PMSF e SDS glycerol stock solution SDS 7 3 wiv Glycerol 29 1 viv Tris base 83 3 mM Bromophenol blue Deionized HO e Tris EDTA solution Tris base 200 mM EDTA 20 mM Deionized HO TCA Laemmli loading buffer Prepare fresh just prior to use SDS glycerol stock solution Tris EDTA solution B mercaptoethanol PMSF Protease inhibitor solution Deionized HO Protocol PT3024 1 Technical Support Version PR7X265 To prepare 10 ml of TCA buffer 200 ulofa 1 M stock solution 66 6 ulof a 7 5 M stock solution 40 ul of a 0 5 M stock solution 500 ul prechille
97. ktail 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 e YPD and appropriate SD liquid medium Recipes in Appendix e 20 and 50 ml culture tubes 014 e Dry ice or liquid nitrogen 1 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 2 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 3 Incubate at 30 C with shaking 220 250 rpm until the ODsoo reaches 0 4 0 6 Depending on the fusion protein this will take 4 8 hr Multiply the ODsoo of a 1 ml sample by the culture volume i e 55 ml to obtain the total number of ODggp units this number will be used in Sections C amp D For example 0 6 x 55 ml 33 total ODsoo units Note D
98. l part of the promoter Ebina et a 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 acZexpression is under control of 1 8 copies of the LexA op Estojak et al 1995 and the minimal GAL7 promoter Because all of the GAL1 UAS sequences have been removed from the lacZ 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 ADH1 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 in 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 ADH1 promoter Table Il At one point it was believed that only this portion was necessary for high le
99. ling 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 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 vvith either dithiothreitol or 2 mercaptoethanol prior to loading the gel Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 17 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 a 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 in a modified transformation to integrate linear
100. ll 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 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 e plating volume 100 ul resuspension volume 0 5 ml e amount of limiting plasmid 0 1 ug 100 cfu x 0 5ml x 1034l ml 5 x 105 cfu ug DNA 100ul x 0 01 x 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 PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 21 Yeast Protocols Handbook CLONTECH Laboratories Inc V
101. m 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 29 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued 22 23 24 25 26 27 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 f the 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 at a time in the luminometer compartment and record the light emission RLU as a5 sec integral Use your blank sample as a reference when interpreting the data Detection using a plate luminometer After Step 21 simply record light signals as 5 sec integrals 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 S
102. 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 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 to disperse the cells Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 41 Yeast Protocols Handbook CLONTECH Laboratories Inc IX Additional Useful Protocols continued c 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 d 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 e Aliquot 20 ul of the cell suspension from Step 3 c into each well of a horizontal row Use a separate rovv for each type of control plasmid f 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 vill spill out of the vvells g 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
103. nce of the medium Note that YPD from CLONTECH already contains glucose Optional For adenine supplemented YPD YPDA prepare YPD medium as above After auto claved medium has cooled to 55 C add 15 ml of a 0 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 Optional For kanamycin containing medium prepare YPD 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 Synthetic dropout SD is a minimal medium used in yeast transformations to select and test for specific phenotypes SD medium is generally prepared by combining a minimal SD base providing a nitrogen base a carbon source and in some cases ammonium sulfate with a stock of dropout solution that contains a specific mixture of amino acids and nucleosides Minimal SD Base and Minimal SD Agar Base either with dextrose glucose or galactose raffinose are available from CLONTECH in convenient powder form See Chapter XI for ordering information If you purchase CLONTECH s Minimal SD Base prepare the medium according to the instructions provided If you prefer you can purchase yeast nitrogen base from another supplier e g Difco 0919 15 3 and prepare SD medium as follows 6 7 g Yeast nitrogen base without amino acids 20 g Agar for plates only 850 ml HO 100 ml o
104. ng CPRG as Substrate Liquid Culture Assay Using a Chemiluminescent Substrate aM OO Go Working with Yeast Plasmids A General Information 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 Hybridization B Generating Yeast Plasmid Segregants C Yeast Mating References MATCHMAKER and Related Products APPENDICES A B C m page 2 Glossary of Technical Terms Yeast Genetic Markers Used in the MATCHMAKER Systems Media Recipes A Yeast Media B E coliMedia Solution Formulations Plasmid Information Yeast Host Strain Information Technical Support TEL 650 424 8222 or 800 662 2566 CLON FAX 650 424 1064 or 800 424 1350 10 12 12 12 14 16 17 18 18 19 20 20 20 22 23 23 25 25 26 27 28 31 31 31 33 36 36 36 39 39 40 41 43 46 47 49 50 50 53 54 58 61 Protocol PT3024 1 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 1 Yeast Promoter Constructs in the MATCHMAKER Cloning Vectors 9 Table 11 Comparison of B galactosidase Assays 24 Table IV Selected Yeast Genes and
105. nsformed 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 D ONPG Appendix D 1 M NaCO Liquid nitrogen page 26 1 Prepare 5 ml overnight cultures in liquid SD selection medium as described in Chapter III A 3 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 mlin 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 induction medium for the strains being assayed 4 Incubate the fresh culture at 30 C for 3 5 hr with shaking 230 250 rpm
106. on is under control of UASg 17 mer xa and the extremely weak minimal promoter of the yeast cytochrome C1 CYC1 gene lacZ under the control of the intact GAL1 promoter can be expressed at 10X the level obtained with the UASg 17 mer say CYC 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 PU69 2A is tightly regulated by the intact GAL2 promoter whose induction properties are similar to those of the GAL7 promoter Reporter genes under the control of a minimal H S3 promoter The native yeast H S3 promoter contains a UAS site recognized by the transcriptional activator GCN4 and two TATA boxes GCN4 regulates one of the TATA boxes TR while the other TATA box TC drives low level constitutive expression of H S3 lyer amp Struhl 1995 TC is not regulated by the native GCN4 binding UAS the GAL7 UAS or artificial VAS constructs Mahadevan amp Struhl 1990 Hope amp Struhl 1986 The H S3 reporter gene in yeast strain Y190 is unusual among the GAL4 two hybrid reporter gene constructs in that it is under the control of the 7 UAS and a minimal promoter containing both HIS3 TATA boxes Flick amp Johnston 1990 The result is high l
107. 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 which differ only physiologically and not in physical form mutant An organism or cell carrying a mutation mutant allele An allele differing from the allele found in the standard or wild type Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 47 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX A Glossary of Technical Terms continued 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 asingle 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 o
108. orking 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 onice vortex vigorously and restreak The glycerol stock tube may be refrozen afew 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 to thoroughly disperse the cells Notes 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 Ifyou 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 When growing overnight cultures of yeast transformants use the appropriate S
109. petent cells or thaw them on ice Chill on ice for 2 min Add 1 ml of LB broth or preferably SOC medium with no antibiotic 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 table top centrifuge Discard supernatant and resuspend pellet in residual liquid Plate cells on appropriate medium LB amp or supplemented M9 Incubate plates at 37 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VII Working With Yeast Plasmids continued 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 107 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 colitransformants a Use a standard plasmid mini prep procedure to isolate plasmid DNA from the E coli transformants Sambrook et al 1989 Notes e If
110. r information on choosing the appropriate SD selection media for particular plasmids host strains and applications Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 11 Yeast Protocols Handbook CLONTECH Laboratories Inc A IV Preparation of Yeast Protein Extracts 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 physical 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 coc
111. r thaw electrocompetent E coli cells Add 1 2 ul of yeast plasmid solution to 40 ul of electrocompetent cells on ice Transfer samples to a prechilled cuvette having a 0 1 cm gap Perform the electroporation according to the manufacturer s instructions Add 1 ml of LB or preferably SOC medium with 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 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 Add 10 ul of yeast plasmid solution to a prechilled Falcon tube Add 100 ul of competent cells to the tube and mix well by gently tapping the tube Incubate on ice for 30 min Heat shock by transferring the tube to a 42 C water bath and incubating for 45 50 sec oo oo SA lt Ss Prepare the chemically com
112. reporter gene in a LexA two hybrid library screening when the reporter gene is maintained on an autonomously replicating plasmid The in vivo assay works for LexA transformants because of the acZ reporter plasmid s high copy number and because of the preamplification step that normally precedes the B 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 galactosidase activity and the K of an interaction Estojak etal 1995 Furthermore quantitative data cannot be compared between different host strains having different acZ 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 l for a discussion of the promoters The liquid ass
113. 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 2 3 Streak replica plate or spread the transformants to be assayed on selection medium containing X gal and BU salts e 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 densi ties to cover a range e g 0 5 x 10 cfu on some 150 mm plates and 2 x 105 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 Incubate plates at 30 C for 4 6 days Check plates every 12 hr up to 96 hr for development of blue color Notes Ifyou 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 Colony lift Filter Assay Reagents and Materials Required Whatman 5 or VWR Grade 410 paper filters sterile Notes 75 mm filters e g VWR 28321 055 can be used with
114. riate SD agar plates 100 mm diameter Notes Prepare the selection media and pour the required number of agar plates in advance 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 Appropriate plasmid DNA in solution check amounts required 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 Sterile PEG LiAc solution Prepare only the volume needed immediately prior to use from 10X stocks Appendix D B 100 DMSO Dimethyl sulfoxide Sigma D 8779 Sterile 1X TE buffer Prepare from 10X TE buffer Appendix D B Sterile glass rod bent Pasteur pipette or 5 mm glass beads for spreading cells on plates Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 19 Yeast Protocols Handbook CLONTECH Laboratories Inc V Yeast Transformation Procedures continued C T
115. round expression f you have an application that requires integration of a plasmid into the yeast genome please see Section V D Transformation 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 see Troubleshooting 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 K1606 1 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 Approp
116. s 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 a cell typically such markers are genes that complement a nutritional requirement or confer resistance to an antibiotic UAS Upstream Activating Sequence yeast DNA sequences that control 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 UASs 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 48 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX B Yeast Genetic Markers Used in the MATCHMAKER Systems TABLE IV SELECTED YEAST GENES AND THEIR ASSOCIATED PHENOTYPES Allele Wildtype Mutant Phenotype of mutant TRP1 tro1 901 Tro 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
117. s be sure that no extra solution is on the outside 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 Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 37 Yeast Protocols Handbook CLONTECH Laboratories Inc VIII Analysis of Yeast Plasmid Inserts by PCR continued c Use a Master Mix To reduce tube to tube variation use a master mix whenever you set up multiple PCR reactions f you wish include the primers in the master mix also f 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 HO instead of DNA template Positive controls are provided with all of CLONTECH s Insert Screening Amplimer Sets Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 38 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc IX Additional Useful Protocols A Yeast Colony Hybridization Yeast colony hybridization is an efficient way to screen a large collection of library transformants forthe presence of an abundant
118. s of the cells If necessary samples can be stored frozen at 20 C If samples have been frozen vortex them again before using them Pour the entire contents of the tube from Step 5 above onto a presoun 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 in TE buffer pH 7 0 Add 200 ul of phenol chloroform isoamyl alchol 25 24 1 Vortex at highest speed for 5 min Centrifuge at 14 000 rpm for 10 min Transfer the aqueous upper phase to a fresh tube Add 8 ul of 10 M ammonium acetate and 500 ul of 95 10096 Ethanol 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 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 e o Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 32 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VII Working With Yeast Plasmids continued C Transforming E coli with Yeast Plasmids We recommend using electroporation Section C 1 when trans
119. stern blot b p LexA pGAD GH ADH1 full length Ethanol repressed High pAS2 1 pAS2 pACT2 pACT ADH1 truncated Constitutive medium pGAD GL ADH1 truncated Constitutive low weak pGAD424 pGAD10 Constitutive very low not detectable pGBT9 pB42AD pGilda GAL 1 full length Repressed by glucose not detectable induced high level by galactose 449 p8op lacZ GAL1 minimal Not regulated by glucose no data or galactose a See Appendix E for vector references b Unpublished data obtained at CLONTECH Laboratories using the appropriate GAL4 domain specific mAb 5398 1 or 25399 1 Soluble protein extracts were prepared from CG 1945 transformed with the indicated plasmid Samples equivalent to 1 ODgo9 unit of cells were electrophoresed and then blotted to nitrocellulose filters The blots were probed with either GAL4 DNA BD mAb 0 5 pg 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 15 000 in TBST 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 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 transforme
120. t al 1993 Optional For eycloheximide containing medium add the appropriate amount of 1 mg ml cyclohex imide 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 50 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc APPENDIX C Media Recipes continued 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 as Y190 and CG 1945 carrying the cyh 2 allele Optional If you wish to add excess adenine to SD medium add 15 ml of 0 2 adenine hemisulfate solution per liter of medium e Pour plates and allow medium to harden at room temperature Store plates inverted in a plastic sleeve at 4 C SD Gal Raf X gal plates Prepare SD medium as described above except use 725 ml of HO 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 d
121. the tube Thoroughly resuspend the cell pellets by vortexing 9 Pool cells in one tube and 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 LiAc 12 Add 0 1 ug of plasmid DNA and 0 1 mg of herring testes carrier DNA to a fresh 1 5 ml tube and mix Notes 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 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 wi
122. ti31HOd3a3t YSANVWHOLVW IIA 318V 1 Protocol PT3024 1 Version PR7X265 TEL 650 424 8222 or 800 662 2566 CLON Technical Support FAX 650 424 1064 or 800 424 1350 page 60 CLONTECH Laboratories Inc Yeast Protocols Handbook ion Informat In Yeast Host Stra APPENDIX F onu suoo s hod l Z29GV ZTV 94 Aq sem ules JOSINDeJd y Jo eueb 0 290 7661 E d holl 9Z4H 10 AHEAH D S GY6L DO SOUEIQI HAMVINHOLV N PEWOJsues old s lHeiqr Ld p Wass 511 u 5 Uu0l1998 8s SE p sn ae Jey 5 5 sedAjouaud slay pue s u B uo uollpuuojul 40 A QEL 88S q s u B y JO si ouuoid ay uo uoneuuoyul 104 4 08 S Gy6 1 0 pue 9 4H 94 Ul eUONOUNjUOU SI u986zSA 7 941 UOHeINW ajqnop si Z 6 219 suonajap e ave suoneinw ggjeb pue peb csiu 1 011 SU e zgza3qv Y1V1z7Tvp SYIzTvD esin 4 4 qyO SYn TV ZSA 1 VOS E vp e6 9661 18 sewer n gen ESIH Q0Z ES 4 ZG EV4N ZLL Zn 106 10 PIVIW s neiq 14 iW2 69f d 1 2091 5siy gape VOS P vple6 3 1 6091X L0G L4A ZL 1 Zn l L06 LAN gape LOL Z PE HL WW 661 TE niq 01 294 108 29 1 00Z SlU ZS un CLYW 3 Hz vx q L ZPIWA 2 1 9 yx v 1 6
123. tor is a cis acting regulatory element derived from E coli For GAL4 based systems either a native GAL UAS or a synthetic UASz 17 mer consensus Sequence 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 generating vector Protocol PT3024 1 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Version PR7X265 FAX 650 424 1064 or 800 424 1350 page 5 Yeast Protocols Handbook CLONTECH Laboratories Inc II Introduction to Yeast Promoters continued TABLE I YEAST PROMOTER CONSTRUCTS USED TO REGULATE REPORTER GENE EXPRESSION IN MATCHMAKER PLASMIDS AND HOST STRAINS Plasmid or Reporter Origin of UAS Origin of Expression levelb host straina gene UAS regulated by TATA sequence Induced uninduced CG 1945 lacZ UASg 17 mer x3 GAL4 CYC1 lovv HIS3 GAL1 GAL4 GAL1 high slightly leaky HF7c lacZ UASg 17 mer x3 GAL4 CYC1 low HIS3 GAL1 GAL4 GAL1 high tight Y190 lacZ GAL1 GAL4 GAL1 high HIS3 GAL1 GAL4 HIS3 TC TR high leaky Y187 lacZ GAL1 GAL4 GAL1 high SFY526 lacZ GAL1 GAL4 GAL1 high PJ69 2A HIS3 GAL1 GAL4 GAL1 high tight ADE2 GAL2 GAL4 GAL2 high tight EGY48 LEU2 LexA opye LexA LEU2 high p8op lacZ la
124. tration To Prepare One Liter Bactotryptone 2 20 g Yeast extract 0 5 5g NaCl 10 mM 10 ml of 1 M NaCl KCI 2 5 mM 2 5 mlof 1 M KCI MgCl 10 mM 10 ml of 1 MMgCl 6 HO MgSO 10 mM 10 ml of 1 M MgSO 7H O Glucose 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 Adjustthe pH to 7 with 5 N NaOH 0 2 ml Adjust the volume to 960 ml with deionized 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 7 0 with 5 N NaOH Autoclave Store broth at 22 C LB amp agar plates Prepare LB broth Sambrook ef 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 colitransformants 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 purchas
125. ts Galacton Star The 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 gt Most sensitive X gal ONPG CPRG X gal Galacton Star in agar plates liquid assay liquid assay filter assay liquid assay Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 24 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc VI B Galactosidase Assays continued B In vivo Plate Assay Using X gal in the Medium Reagents and Materials Required Appropriate SD agar plates containing X gal 80 mg L and 1X BU salts Appendix 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 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
126. two hybrid systems Curr Opinion in Biotechnology 5 482 486 Other References Alexandre C Grueneberg D A amp Gilman M Z 1993 Studying Heterologous Transcription Factors in Yeast 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 PCR amplification of up to 35 kb DNA with high fidelity and high yield from 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 8 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 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 F and cytoplasmic domains of CD4 and CD8 alpha in yeast Eur J Immunol 25 240
127. ulturing and handling yeast e information on the yeast promoters used in the MATCHMAKER Systems two protocols for preparing protein extracts from yeast e quantitative and qualitative B galactosidase assays for use with lacZ yeast reporter strains 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 asmall scale lithium acetate yeast transformation protocol 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 K1604 1 K1605 1 and LexA Two Hybrid System 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 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 The MATCHMAKER Libraries are constructed in vectors that express inserts as fusions to a transcriptional activation domain and are thus a convenient resource for r
128. ura A 1983 Transformation of intact yeast cells treated with alkali cations J Bacteriol 153 163 168 Iwabuchi 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 1 425 1436 Johnston M Flick J S amp Pexton T 1994 Multiple mechanisms provide rapid and stringent glucose 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 1 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 Li B amp Fields S 1993 Identification of mutations in p53 that affect its binding to SV40 T 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 D
129. uring late log phase the ADH1 promoter shuts down and the level of endogenous yeast proteases increases 4 Quickly chill the culture by pouring it into a prechilled 100 ml centrifuge tube halfway filled with ice 5 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 7 Recover the pellet by centrifugation at 1 000 x g for 5 min at 4 C 8 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 12 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc 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 Vortex vigorously for 1 min e Centrifuge at 14 000 rpm for 5 min 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 e Combine with supernatant second supernatant e Place on ice Combined supernatants Immediately load gel or freeze at 70
130. uspend pellets in the residual liquid total volume 50 ul 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 of the 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 a centrifuge 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 Add 10 wl of lyticase solution to each tube Thoroughly resuspend the cells by vortexing or repeatedly pipetting up and down 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 Add 10 ul of 20 SDS to each tube and vortex vigorously for 1 min to mix Put the samples through one freeze thaw cycle at 20 C and vortex again to ensure complete lysi
131. vel 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 et al 1991 Ruohonen etal 1995 Tornow amp Santangelo 1990 This observation has been confirmed at CLONTECH by quantitative Western 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 AD cloning vector pB42AD and the alternative DNA BD vector pGilda used in the MATCHMAKER LexA Two Hybrid 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 Technical Support TEL 650 424 8222 or 800 662 2566 CLON Protocol PT3024 1 page 8 FAX 650 424 1064 or 800 424 1350 Version PR7X265 Yeast Protocols Handbook CLONTECH Laboratories Inc ll Introduction to Yeast Promoters continued TABLE Il YEAST PROMOTER CONSTRUCTS IN THE MATCHMAKER CLONING VECTORS Regulation Signal Relative Protein Strength on Vectorsa Promoter Expression Level We
132. 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 A See Sambrook et al 1198911 1 22 1 23 e Boiling lysis is not recommended for isolation of plasmids from endA bacteria If you are using a commercial plasmid preparation kit follow the manufacturer s directions for host strains that are endAt 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 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 Alul or Haelll 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 If you performed a nutritional selection on M9 minimal medium repeat the transforma tion 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 ver
133. ys 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 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 CYHS2 gene is dominant to the cyh 2 mutant allele Thus when transformed with a plasmid such as pAS2 1 that contains the wild type 2 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 c

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