pYES2/pYES3
Contents
1. Shipping and p YES2 CT pYES3 and pYC2 vectors are shipped on wet ice Upon receipt store Storage vectors at 20 C Kit Contents All vectors are supplied as detailed below Store the vectors at 20 C Cat no Vector Composition Amount 40 uL of 0 5 ug uL vector in 10 mM Tris HCI PENE 1 mM EDTA pH 8 0 20 pg nn 40 uL of 0 5 ug uL in 10 mM Tris HCl uL of 0 5 ug uL vector in 10 mM Tris j pYES2 CT lacZ 1 mM EDTA pH 8 0 20 ug 40 uL of 0 5 ug uL vector in 10 mM Tris HCI presion 1 mM EDTA pH 8 0 20 08 ve 40 uL of 0 5 ug uL in 10 mM Tris HCl uL of 0 5 ug uL vector in 10 mM Tris pYES3 CT lacZ 1 mM EDTA pH 8 0 20 ug 40 uL of 0 5 ug uL vector in 10 mM Tris HCI Be 1 mM EDTA pH 8 0 20 08 ae 40 uL of 0 5 ug uL in 10 mM Tris HCI uL of 0 5 ug uL vector in 10 mM Tris y pYC2 CT lacZ 1 mM EDTA pH 8 0 20 ug The INVSc1 Yeast Strain is included with each vector kit Note Note For long term storage of your stab we recommend preparing a glycerol stock immediately upon receipt and storing at 80 C Genotype The genotype and phenotype of the INVSc1 host strain are provided below nu of Genotype MATa his3A1 leu2 trp1 289 ura3 52 MATa his3A1 leu2 trp1 289 ura3 52 c Preparing INVSc1 Glycerol Stocks Intended Use Phenotype His Leu Trp Ura We recommend that you prepare a set of glycerol master stocks within two weeks of receiving the INVSc1 yeast cells 1 Use a sterile loop to inoculate a 50 mL tube c2. This product is the subject of one or more of U S Patent Nos 5 618 676 5 854 018 5 856 123 5 919 651 and foreign equivalents Rights to use this product are limited to academic research use only Non academic entities are required to obtain a separate license from Washington Research Foundation to utilize this product for any use Washington Research Foundation 2815 Eastlake Avenue East Suite 300 Seattle Washington 98102 Tel 206 336 5600 Fax 206 336 5615 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Bouton A H and Smith M M 1986 Fine Structure Analysis of the DNA Sequence Requirements for Autonomous Replication of Saccharomyces cerevisiae Plasmids Mol Cell Biol 6 2354 2363 Cigan A M and Donahue T F 1987 Sequence and Structural Features Associated with Translational Initiator Regions in Yeast A Review Gene 59 1 18 Gietz D Jean A S Woods R A and Schiestl R H 1992 Improved Method for High Efficiency Transformation of Intact Yeast Cells Nuc Acids Res 20 1425 Gietz R D Schiestl R H Willems A R and Woods R A 1995 Studies on the Transformation of Intact Yeast Cells by the LiAc SS DNA PEG Procedure Yeast 11 355 360 Giniger E Barnum S M and Ptashne M 1985 Specific DNA B
3. Oo S I Sac T7 promoter priming site bases 475 494 Multiple cloning site bases 501 588 V5 epitope bases 607 648 Polyhistidine 6xHis region bases 658 675 CYC1 transcription termination signal bases 708 961 CYC1 reverse priming site bases 725 743 pUC origin bases 1145 1818 Ampicillin resistance gene bases 1963 2823 complementary strand TRP1 promoter bases 3031 3132 TRP1 gene bases 3133 3807 2u origin bases 3859 5330 f1 origin bases 5398 5853 complementary strand 16 Continued on next page pYES3 CT Vector Continued p YES3 CT 5870 bp contains the following elements All features have been functionally tested Feature Benefit GAL1 promoter Permits galactose inducible expression of genes cloned into pYES3 CT West et al 1984 GALI forward priming site Allows sequencing through the insert T7 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert Multiple cloning site with 8 unique sites and two BstX I sites Allows insertion of your gene and facilitates cloning in frame with the V5 epitope and the polyhistidine tag V5 epitope Permits detection of the fusion protein with the Anti V5 Antibody or the Anti V5 HRP Antibody Southern et al 1991 C terminal polyhistidine 6xHis tag Permits purification of your fusion protein on metal chelating resin such as ProBond I
4. You may verify the size of the DNA on a gel Aliquot the sonicated DNA into four 50 mL conical centrifuge tubes 25 mL per tube Extract with 25 mL of TE saturated phenol Centrifuge at 10 000 x g for 5 minutes at 4 C Transfer the DNA upper layer to a fresh 50 mL conical centrifuge tube Extract with 25 mL of TE saturated phenol chloroform isoamyl alcohol 25 24 1 Centrifuge at 10 000 x g for 5 minutes at 4 C Transfer the DNA upper layer to a fresh 50 mL conical centrifuge tube Extract with 25 mL of chloroform Centrifuge at 10 000 x g for 5 minutes at 4 C Transfer the DNA upper layer to a 250 mL centrifuge bottle Add 5 mL of 3 M sodium acetate pH 6 0 1 10 volume and 125 mL ice cold 20 C 95 ethanol 2 5 volume to precipitate DNA Pellet the DNA at 12 000 x g for 15 minutes at 4 C Wash the DNA once with 200 mL 70 ethanol and centrifuge as described in step 9 Partially dry DNA by air or in a Speed Vac cover tubes with parafilm and poke holes in top for 20 minutes Transfer DNA to a 250 mL sterile flask Dissolve DNA in 100 mL sterile TE 10 mg mL Boil for 20 minutes to denature DNA Immediately place on ice aliquot in 1 mL samples and freeze at 20 C Accessory Products Introduction The following products may be used with the pYES2 CT pYES3 and pYC2 vectors For details visit www invitrogen com or contact Technical Support see page29 Ite
5. 0 4 8 12 16 and 24 hours after addition of cells to the induction medium For each time point remove 5 mL of culture from the flask and determine the OD o of each sample You will use this information when assaying for your recombinant fusion protein see Step 3 on the next page Centrifuge the cells at 1 500 x g for 5 minutes at 4 C Decant the supernatant Resuspend cells in 500 uL of sterile water Transfer cells to a sterile microcentrifuge tube Centrifuge samples for 30 seconds at top speed in the microcentrifuge 9 Remove the supernatant 10 Store the cell pellets at 80 C until ready to use Proceed to the next section to prepare cell lysates to detect your recombinant protein see the next page Continued on next page 10 Expression of Recombinant Protein Continued Detecting To detect expression of your recombinant fusion protein by western blot see Recombinant below you may use the Anti V5 antibodies or the Anti His C term antibodies Fusion Protein available from Invitrogen see page 27 for ordering information or an antibody to your protein of interest You will also need to prepare a cell lysate from your yeast transformant A general protocol for small scale preparation of cell lysates using acid washed glass beads is provided below for your convenience Other protocols are suitable Refer to Current Protocols in Molecular Biology Ausubel et al 1994 for more information For large scale prepar
6. BamH E X Comments for pYES3 CT lacZ 8878 nucleotides GALT promoter bases 1 451 GAL1 forward priming site bases 414 437 T7 promoter priming site bases 475 494 LacZ ORF bases 528 3583 V5 epitope bases 3615 3656 Polyhistidine 6xHis region bases 3666 3683 CYC1 transcription termination signal bases 3716 3969 CYC1 reverse priming site bases 3733 3751 pUC origin bases 4153 4826 Ampicillin resistance gene bases 4971 5831 complementary strand TRP1 promoter bases 6039 6140 TRP1 gene bases 6141 6815 2u origin bases 6867 8338 f1 origin bases 8406 8861 complementary strand pYC2 CT Vector Map of pYC2 CT Comments for pYC2 CT 4627 nucleotides GAL1 promoter bases 1 451 GAL1 forward priming site bases 414 437 The figure below summarizes the features of the pYC2 CT vector The vector sequence for pYC2 CT is available for downloading from www invitrogen com or from Technical Support see page 29 Asp718 Kpn I BamH I BstX I EcoR I BstX I Pme I V5 epitope 6xHis stop Not I Xho I Xba I ne E Sac T7 promoter priming site bases 475 494 Multiple cloning site bases 501 594 V5 epitope bases 607 648 V5 C term reverse priming site bases 616 636 Polyhistidine 6xHis region bases 658 675 CYC1 transcription termination signal bases 708 961 CYC1 reverse priming site bases 725 743 pUC origin bases 1145 1818 complementary strand Ampicillin resistance gene bas
7. custom primer synthesis service Visit www invitrogen com for more details Invitrogen has a wide variety of yeast expression YES vectors utilizing the GAL1 promoter Vectors are available with the Xpress N terminal or V5 C terminal epitope for detection the 2u origin or CEN6 ARSH4 sequence for high copy or low copy replication and either dominant or auxotrophic markers for selection in yeast All vectors contain a polyhistidine tag for purification of recombinant protein using ProBond resin For more information on the YES expression vectors available see www invitrogen com or call Technical Support see page 29 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Fre
8. hours after galactose induction Recombinant fusion protein can be detected in cells that have been cultured in raffinose by 2 hours after galactose induction If you are assaying for expression of your recombinant fusion protein for the first time we recommend that you perform a time course to optimize expression of your recombinant protein e g 0 4 8 12 16 24 hours after galactose induction A standard protocol is provided below to perform a time course experiment Other protocols are suitable 1 Inoculate a single colony of INVSc1 containing your pYES2 CT pYES3 CT or pYC2 CT construct into 15 mL of the appropriate SC selective medium containing 2 glucose or 2 raffinose Grow overnight at 30 C with shaking 2 Determine the ODs of your overnight culture Calculate the amount of overnight culture necessary to obtain an ODs of 0 4 in 50 mL of induction medium SC selective medium containing 2 galactose Example Assume that the ODs of an overnight culture is 3 OD o0 per mL Then the amount of overnight culture needed to inoculate a 50 mL culture to OD600 0 4 is 0 4 OD mL 50 mL 6 67 mL 3 OD mL 3 Remove the amount of overnight culture as determined in Step 2 and pellet the cells at 1 500 x g for 5 minutes at room temperature Discard the supernatant 4 Resuspend the cells in 50 mL of induction medium See page 22 for a recipe for induction medium Grow at 30 C with shaking 5 Harvest an aliquot of cells at
9. in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 29 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 141 Expression of Polypeptides in Yeast 30 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany
10. note that there are two BstX I sites in the polylinker The Xba I site is not unique in pYES3 CT Continued on next page Cloning into pYES2 CT pYES3 CT or pYC2 CT Continued E coli Transformation EN Y A RECO Nor N Preparing a Glycerol Stock Plasmid Preparation Transform your ligation mixtures into a competent recA endA E coli strain of your choice Select for transformants on LB plates containing 50 to 100 pg mL ampicillin Select 10 20 clones and analyze by restriction digest or sequencing for the presence and orientation of your insert We recommend that you sequence your construct to confirm that your gene is fused in frame with the C terminal V5 epitope and the polyhistidine 6xHis tag To sequence your construct we suggest using either the GAL1 Forward or the T7 Promoter primer sequences along with the CYC1 Reverse primer sequences Refer to the diagram on the previous page for the sequences and location of the priming sites Once you have identified the correct clone be sure to purify the colony and make a glycerol stock for long term storage It is also a good idea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony out on an LB plate containing 50 pg mL ampicillin Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 mL of LB containing 50 pg mL ampicillin 3 Grow the culture to mid log phase ODso 0 5 0 7 Mix 0 85 mL o
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12. 49 6956 complementary strand CEN6 ARSH4 bases 6969 7487 21 Recipes SC Minimal Medium and Plates Induction Medium 22 SC is synthetic minimal defined medium for yeast 0 67 yeast nitrogen base without amino acids but with ammonium sulfate 2 carbon source i e glucose or raffinose 0 01 adenine arginine cysteine leucine lysine threonine tryptophan uracil 0 005 aspartic acid histidine isoleucine methionine phenylalanine proline serine tyrosine valine 2 agar for plates 1 Dissolve the following reagents in 900 mL deionized water 800 mL if preparing medium containing raffinose Note We make medium and plates as we need them and weigh out each amino acid Many researchers prepare 100X solutions of each amino acid that they need Reminder Omit uracil to make selective plates for growing pYES2 CT or pYC2 CT transformants Omit tryptophan to make selective plates for growing pYES3 CT transformants 6 7 g Yeast Nitrogen Base 0 1 g each 0 05 g each adenine aspartic acid arginine histidine cysteine isoleucine leucine methionine lysine phenylalanine threonine proline tryptophan W serine uracil U tyrosine valine If you are making plates add the agar after dissolving the reagents above Autoclave at 15 psi 121 C for 20 minutes Cool to 50 C and add 100 mL of filter sterilized 20 glucose or 200 mL of filter sterilized 10 raffinose 4 C Plates are stabl
13. CT Note that B galactosidase will be expressed as a fusion protein containing the C terminal V5 epitope and a polyhistidine 6xHis tag Map of The figure below summarizes the features of the pYES2 CT lacZ vector The pYES2 CT lacZ vector nucleotide sequence for pYES2 CT lacZ is available for downloading from www invitrogen com or by contacting Technical Support see page 29 Pme V5 epitope 6XHis stop Comments for pYES2 CT lacZ 8971 nucleotides GAL1 promoter bases 1 451 GAL1 forward priming site bases 414 437 T7 promoter priming site bases 475 494 LacZ ORF bases 528 3583 V5 epitope bases 3615 3656 Polyhistidine 6xHis region bases 3666 3683 CYC1 transcription termination signal bases 3716 3969 CYC1 reverse priming site bases 3733 3751 pUC origin bases 4153 4826 Ampicillin resistance gene bases 4971 5831 complementary strand URA3 gene bases 5849 6956 complementary strand 2u Origin bases 6960 8431 f1 origin bases 8499 8954 complementary strand 15 pYES3 CT Vector Map of pYES3 CT Comments for pYES3 CT 5870 nucleotides GAL1 promoter bases 1 451 GAL 1 forward priming site bases 414 437 The figure below summarizes the features of the pYES3 CT lacZ vector The vector sequence for pYES3 CT lacZ is available for downloading from www invitrogen com or from Technical Support see page 29 Asp718 I Kpn BamH BstX EcoR BstX Pme V5 epitope 6xHis stop Not Xho
14. TE Incubate the cells at room temperature for 10 minutes For each transformation mix together 1 ug plasmid DNA and 100 ug denatured sheared salmon sperm DNA with 100 uL of the yeast suspension from Step 5 Add 700 uL of 1X LiAc 40 PEG 3350 1X TE and mix well Incubate solution at 30 C for 30 minutes Add 88 uL DMSO mix well and heat shock at 42 C for 7 minutes 10 Centrifuge in a microcentrifuge for 10 seconds and remove supernatant 11 Resuspend the cell pellet in 1 mL 1X TE and re pellet 12 Resuspend the cell pellet in 50 100 uL 1X TE and plate on a selective plate To calculate the number of yeast cells assume that 1 OD w unit 2 0 x 10 yeast cells Note 25 Preparing Denatured Salmon Sperm DNA Materials Needed Protocol 26 Salmon Sperm DNA Sigma Aldrich Catalog no D1626 1X TE Sonicator 50 mL conical centrifuge tubes TE saturated phenol TE saturated phenol chloroform isoamyl alcohol 25 24 1 Chloroform Low speed centrifuge 3 M sodium acetate pH 6 0 95 ethanol 20 C 250 mL centrifuge bottle Boiling water bath 10 11 12 13 In a 250 mL flask dissolve 1 g salmon sperm DNA into 100 mL of TE 10 mg mL Pipet up and down with a 10 mL pipette to dissolve completely Incubate overnight at 4 C on a rotating wheel Using a sonicator with a large probe sonicate the DNA twice for 30 seconds at 3 4 power The resulting DNA will have an average size of 7 kb
15. alysis or functional assay 7 Use metal chelating resin such as ProBond to purify your recombinant protein Methods Cloning into pYES2 CT pYES3 CT or pYC2 CT General Molecular For help with DNA ligations E coli transformations restriction enzyme analysis Biology DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Techniques Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 E coli Strain Many E coli strains are suitable for the propagation of pYES2 CT pYES3 CT and pYC2 CT We recommend that you propagate the vectors in E coli strains that are recombination deficient recA and endonuclease deficient end A For your convenience TOP10 E coli are available as chemically competent or electrocompetent cells from Invitrogen page 27 Transformation You may use any method of your choice for transformation Chemical Method transformation is the most convenient for most researchers Electroporation is the most efficient and the method of choice for large plasmids Propagating and To propagate and maintain the pYES2 CT pYES3 CT and pYC2 CT vectors Maintaining use a small amount of the supplied 0 5 ug uL stock solution in TE pH 8 0 to Plasmids transform a recA endA E coli strain like TOP10F DH5a JM109 or equivalent Select transformants on LB plates containing 50 100 pg mL ampicillin Be sure to prepare a glycerol stock
16. ations culture volumes over 1 liter see Scale up on the next page Materials Needed e Breaking buffer 50 mM sodium phosphate pH 7 4 see page 23 for recipe of stock buffer 1 mM EDTA omit EDTA if using this buffer for purification on metal chelating resins 5 glycerol 1 mM PMSF e Acid washed glass beads 0 4 0 6 mm size Sigma Aldrich Catalog no G8772 Protocol 1 You may prepare cell lysates from frozen cells or fresh cells Reminder You will need to know the ODso of your cell sample s before beginning see Step 5 previous page 2 Resuspend fresh or frozen cell pellets in 500 uL of breaking buffer Centrifuge at 1 500 x g for 5 minutes at 4 C to pellet cells 3 Remove supernatant and resuspend the cells in a volume of breaking buffer to obtain an ODeo of 50 100 Use the ODs determined in Step 5 previous page to calculate the appropriate volume of breaking buffer to use Add an equal volume of acid washed glass beads Vortex mixture for 30 seconds followed by 30 seconds on ice Repeat four times for a total of four minutes to lyse the cells Cells will be lysed by shear force You can check for the extent of lysis by checking a small aliquot under the microscope Centrifuge in a microcentrifuge for 10 minutes at maximum speed Remove supernatant and transfer to a fresh microcentrifuge tube Assay the lysate for protein concentration using BSA as a standard 8 Add SDS PAGE sample buffer to a final concent
17. d high copy replication in E coli Ampicillin resistance gene Permits selection of transformants in E coli URA3 gene Permits selection of yeast transformants in uracil deficient medium CEN6 ARSH4 sequence Permits non integrative centromeric maintenance and low copy replication in yeast Sikorski and Hieter 1989 pYC2 CT lacZ Description pYC2 CT lacZ is a 7635 bp control vector containing the gene for B galactosidase This vector was constructed by ligating a 3 1 kb fragment containing the lacZ gene into the BamH I Not I site of pYC2 CT Note that B galactosidase will be expressed as a fusion protein containing the C terminal V5 epitope and a polyhistidine 6xHis tag Map of The figure below summarizes the features of the pYC2 CT lacZ vector The pYC2 CT lacZ vector nucleotide sequence for pYC2 CT lacZ is available for downloading from www invitrogen com or from Technical Support see page 29 V5 epitope 6xHis stop Comments for pYC2 CTllacZ 7635 nucleotides GAL1 promoter bases 1 451 GAL1 forward priming site bases 414 437 T7 promoter priming site bases 475 494 LacZ ORF bases 528 3583 V5 epitope bases 3615 3656 Polyhistidine 6xHis region bases 3666 3683 CYC1 transcription termination signal bases 3716 3969 CYC1 reverse priming site bases 3733 3751 pUC origin bases 4153 4826 Ampicillin resistance gene bases 4971 5831 complementary strand URA3 gene bases 58
18. e 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech supportQinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Certificate of Analysis Limited Warranty Safety Data Sheets MSDSs are available on our website at www invitrogen com sds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored
19. e for 6 months Pour plates and allow the plates to harden Invert the plates and store at If you are making induction medium follow Steps 1 3 above except dissolve the reagents in 800 mL of deionized water Cool the medium to 50 C and add 100 mL of filter sterilized 20 galactose and 100 mL of filter sterilized 10 raffinose to the medium Continued on next page Recipes Continued Important YPD 0 1 M Sodium Phosphate pH 7 4 When making stock solutions of raffinose do not autoclave the stock solution Autoclaving the solution will convert the raffinose to glucose Filter sterilize the stock solution Yeast Extract Peptone Dextrose Medium 1 liter 1 yeast extract 2 peptone 2 dextrose D glucose 1 Dissolve the following in 1000 mL of water 10 g yeast extract 20 g peptone 20 g dextrose see note below if making plates Optional Add 20 g agar if making plates Autoclave for 20 minutes on liquid cycle Store medium at room temperature or cool the medium and pour plates The shelf life is approximately one to two months Note If making plates omit dextrose from Step 1 Autoclaving agar and dextrose together will cause the dextrose to caramelize Prepare a separate stock solution of 20 dextrose and autoclave or filter sterilize After the YPD broth 900 mL volume has been autoclaved add 100 mL of 20 dextrose to the medium Materials Needed Sodium phosphate monobasic NaH PO H 0 S
20. es 1963 2823 complementary strand URA3 gene bases 2841 3948 complementary strand CENG ARSHA4 bases 3961 4479 Continued on next page 19 pYC2 CT Vector Continued Features of pYC2 CT 20 pYC2 CT 4627 bp contains the following elements All features have been functionally tested Feature Benefit GAL1 promoter Permits galactose inducible expression of genes cloned into pYC2 CT West et al 1984 GALI forward priming site Allows sequencing through the insert T7 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert Multiple cloning site with 9 unique sites and two BstX I sites Allows insertion of your gene and facilitates cloning in frame with the V5 epitope and the polyhistidine tag V5 epitope Permits detection of the fusion protein with the Anti V5 Antibody or the Anti V5 HRP Antibody Southern et al 1991 C terminal polyhistidine 6xHis tag Permits purification of your fusion protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody and the Anti His C term HRP Antibody Lindner et al 1997 CYC1 transcription termination signal Permits efficient termination and stabilization of mRNA CYC1 reverse priming site Allows sequencing through the insert pUC origin Allows maintenance an
21. eu2 trp1 289 trp1 289 ura3 52 ura3 52 Phenotype His Lew Trp Ura Note that INVSc1 is a diploid strain that is auxotrophic for histidine leucine tryptophan and uracil The strain will not grow in SC minimal medium that is deficient in histidine leucine tryptophan or uracil A recipe for preparation of SC minimal medium is provided in the Appendix page 22 Important The INVSc1 strain is a suitable strain to use for expression purposes but should not be used for genetic analyses because it does not sporulate well To initiate a culture of INVSc1 from the stab provided with the kit streak a small amount from the stab on a YPD plate see Appendix for recipe page 23 and incubate at 30 C Once growth is established you may check the phenotype of the strain by streaking a single colony on an SC minimal plate supplemented with the appropriate amino acids INVSc1 will not grow in SC minimal medium that is deficient in histidine leucine tryptophan or uracil Be sure to make glycerol stocks of the strain Store glycerol stocks at 80 C If you plan to use the strain directly from plates be sure that the plates are less than 4 days old The pYES2 CT pYES3 CT and pYC2 CT vectors are supplied with a corresponding positive control vector pYES2 CT lacZ pYES3 CT lacZ and pYC2 CT lacZ respectively to help you optimize expression conditions for your protein The gene encoding B galactosidase is expressed in yeast cells under
22. f culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C You may use any method of your choice to prepare purified plasmid DNA for small scale yeast transformation Standard protocols can be found in Current Protocols in Molecular Biology Ausubel et al 1994 or Molecular Cloning A Laboratory Manual Sambrook et al 1989 If you need ultrapure DNA for sequencing we recommend isolating plasmid DNA using the PureLink HiPure Plasmid Miniprep Kit or the PureLink HiPure Plasmid Midiprep Kit see page 27 Yeast Transformation Introduction Basic Yeast Molecular Biology Genotype Phenotype of INVSc1 Initiating INVSc1 Culture Positive Control In this section you will use a small scale yeast transformation protocol to transform your pYES2 CT pYES3 CT or pYC2 CT construct into the INVSc1 yeast host strain included with each vector After transformation expression of your recombinant fusion protein from pYES2 CT pYES3 CT or pYC2 CT can be induced using galactose To familiarize yourself with basic yeast molecular biology and microbiological techniques refer to Current Protocols in Molecular Biology Unit 13 Ausubel et al 1994 and the Guide to Yeast Genetics and Molecular Biology Guthrie and Fink 1991 for information on preparing yeast media and handling yeast The genotype and phenotype of the INVSc1 host strain are provided below Genotype his3A1 his3A1 leu2 l
23. he manufacturer s recommendations Appendix pYES2 CT Vector Map of pYES2 CT The figure below summarizes the features of the pYES2 CT vector The vector sequence for pYES2 CT is available for downloading from www invitrogen com or from Technical Support see page 29 Asp718 I Kpn I BamH I BstXx EcoR I BstX V5 epitope 6xHis stop Not I Xho I Xba I ne E Sac Pme Comments for pYES2 CT 5963 nucleotides GAL1 promoter bases 1 451 GAL1 forward priming site bases 414 437 T7 promoter priming site bases 475 494 Multiple cloning site bases 501 594 V5 epitope bases 607 648 Polyhistidine 6xHis region bases 658 675 CYC1 transcription termination signal bases 708 961 CYC1 reverse priming site bases 725 743 pUC origin bases 1145 1818 Ampicillin resistance gene bases 1963 2823 complementary strand URA3 gene bases 2841 3948 complementary strand 2u origin bases 3952 5423 f1 origin bases 5491 5946 complementary strand Continued on next page 13 pYES2 CT Vector Continued Features of pYES2 CT 14 pYES2 CT 5963 bp contains the following elements All features have been functionally tested Feature Benefit GAL1 promoter Permits galactose inducible expression of genes cloned into pYES2 CT West et al 1984 GALI forward priming site Allows sequencing through the insert T7 promoter priming site Allows for in vitro transcription in
24. igma Aldrich 59638 Sodium phosphate dibasic Na2HPO 7H2O Sigma Aldrich 59390 1 Prepare 100 mL of 1 M NaH2PO H20 by dissolving 13 8 g in 90 mL of deionized water Bring volume up to 100 mL Filter sterilize 2 Prepare 100 mL of 1 M Na HPO 7H 0O by dissolving 26 81 g in 90 mL of deionized water Bring volume up to 100 mL Filter sterilize 3 For 1 liter of 0 1 M sodium phosphate pH 7 4 mix together 22 6 mL of 1M NaH PO and 77 4 mL of 1 M Na HPO Bring the volume up to 1 liter with sterile water 4 Filter sterilize and store at room temperature Continued on next page 23 Recipes Continued 10X TE 1X TE 10X LiAc 1X LiAc 1X LiAc 0 5X TE 1X LiAc 40 PEG 3350 1X TE 24 100 mM Tris pH 7 5 10 mM EDTA 1 For 100 mL dissolve 1 21 g of Tris base and 0 37 g of EDTA in 90 mL of deionized water 2 Adjust the pH to 7 5 with concentrated HCl and bring the volume up to 100 mL 3 Filter sterilize and store at room temperature Alternatively you can make the solution using 1 M Tris HCI pH 7 5 and 0 5 M EDTA pH 8 0 10 mM Tris pH 7 5 1 mM EDTA Dilute 10X TE 10 fold with sterile water 1 M Lithium Acetate pH 7 5 1 For 100 mL dissolve 10 2 g of lithium acetate in 90 mL of deionized water 2 Adjust pH to 7 5 with dilute glacial acetic acid and bring up the volume to 100 mL 3 Filter sterilize and store at room temperature 100 mM Lithium Acetate pH 7 5 Dilute 10X LiAc
25. inding of GAL4 a Positive Regulatory Protein of Yeast Cell 40 767 774 Guthrie C and Fink G R 1991 Guide to Yeast Genetics and Molecular Biology In Methods in Enzymology Vol 194 J N Abelson and M I Simon eds Academic Press San Diego CA Hartley J L and Donelson J E 1980 Nucleotide Sequence of the Yeast Plasmid Nature 286 860 865 Hill J Donald K A and Griffiths D E 1991 DMSO Enhanced Whole Cell Yeast Transformation Nuc Acids Res 19 5791 Johnston M and Davis R W 1984 Sequences that Regulate the Divergent GAL1 GAL10 Promoter in Saccharomyces cerevisiae Mol Cell Biol 4 1440 1448 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Panzeri L and Philippsen P 1982 Centromeric DNA from Chromosome VI in Saccharomyces cerevisiae Strains EMBO J 1 1605 1611 Romanos M A Scorer C A and Clare J J 1992 Foreign Gene Expression in Yeast A Review Yeast 8 423 488 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Pres
26. invitrogen pYES2 CT pYES3 CT and pYC2 CT Yeast expression vectors with C terminal tags and auxotrophic selection markers Catalog no V8251 20 V8253 20 and V8255 20 Rev date 30 November 2009 Manual part no 25 0304 MAN0000128 ii Table of Contents Kit Contents and Stora gE sr srsti eia iera a AEE RA AN EAE AE ESETE E E ERES SETE E iv Lg lla o 8 oz 4 La y EA e dende 1 TS A A TN 1 Method A 3 Cloning into pYES2 CT pYES3 CT or pYC2 CT eisini eresse inhibisies iea S 3 Yeast Transformations essnee nnen ek hs isdn 7 Expression of Recombinant Protein svrvsrrvrrererererrrrrrrrrrrnenevrvsvsrravevevevenensrsrsenevenensrsenrsrnenensvssrssanenenene 9 APP TK ee 13 PYESZ CT VEG uante irene a BB lan 13 PYES2JET NCZ eiere dier Ge hardere dets bbt 15 PYESSLET VECTOR a SAG 16 PYES3 CT lacZ ass dio 18 PYC2 CT Vectors a nad ad till till iria 19 PYE2 CT lacZ isaac ali piles seman tod 21 E RN RA 22 Small Scale Yeast Transformation messervnvervrvrrnrerraversrvrrerervaversrvrsesesvavassrvssesessrvrsesesvevarsrvssesessevanssvsnenee 25 Preparing Denatured Salmon Sperm DNA rserrnrnsnveveveverenrsrsrsrsrnenensvsvsrraveverevevenensnsrsrsenenensnsesesrnenenenensn 26 Accessory Products Bar RA SS Feen 27 Technical Supports caicioosaononcio ninos Senna aRt aR Lasts coat ae ns ehididen eenaa BEENS np cgnaesehdassdaaeae dha alet 29 Purchaser Notifica vomita aa 30 A O 31 iii Kit Contents and Storage
27. m Amount Catalog no 6 x 2 mL precharged prepacked K850 01 ProBond Purification ProBond resin columns and System buffers for native and denaturing purification F 50 mL R801 01 ProBond Resin 150 mL R801 15 Electrocomp TOP10F 5 x 80 uL C665 55 One Shot TOP10F Chemically Competent E coli SH 2000 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 B Gal Assay Kit 80 mL K1455 01 B Gal Staining Kit 1 kit K1465 01 S c EasyComp Kit 1 kit K5050 01 Antibodies If you do not have an antibody specific to your protein Invitrogen offers the Anti V5 or Anti His C term antibodies to detect your recombinant fusion protein Horseradish peroxidase HRP and alkaline phosphatase AP conjugated antibodies are available for convenient one step detection Antibody Epitope Catalog no Anti V5 Detects a 14 amino acid epitope R960 25 Anti V5 HRP derived from the P and V proteins of R961 25 the paramyxovirus SV5 Southern et Anti V5 AP N R962 25 GKPIPNPLLGLDST Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP tag requires the free carboxyl group R931 25 for detection Lindner et al 1997 Anti His C term AP HHHHHH COOH R932 25 Continued on next page 27 Accessory Products Continued Primers Other Yeast Expression Vectors 28 For your convenience Invitrogen offers a
28. n addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody and the Anti His C term HRP Antibody Lindner et al 1997 CYC1 transcription termination signal Permits efficient termination and stabilization of mRNA CYC1 reverse priming site Allows sequencing through the insert pUC origin Allows maintenance and high copy replication in E coli Ampicillin resistance gene Permits selection of transformants in E coli TRP1 promoter Allows expression of the TRP1 gene TRP1 gene Permits selection of yeast transformants in tryptophan deficient medium Tschumper and Carbon 1980 2p origin Permits episomal maintenance and high copy replication in yeast fl origin Allows rescue of single stranded DNA 17 pYES3 CT lacZ Description pYES3 CT lacZ is a 8878 bp control vector containing the gene for Map of B galactosidase This vector was constructed by ligating a 3 1 kb fragment containing the lacZ gene into the BamH I Not I site of pYES3 CT Note that B galactosidase will be expressed as a fusion protein containing the C terminal V5 epitope and a polyhistidine 6xHis tag The figure below summarizes the features of the pYES3 CT lacZ vector The pYES3 CT lacZ vector nucleotide sequence for pYES3 CT lacZ is available for downloading 18 from www invitrogen com or by contacting Technical Support see page 29 Hind I Asp718
29. of each plasmid for long term storage see page 6 24 Origin The pYES2 CT and pYES3 CT vectors contain the 2u origin for maintenance and replication in yeast The sequence containing the 2u origin was originally isolated from the naturally occurring yeast 2u plasmid Hartley and Donelson 1980 When placed in a heterologous expression plasmid i e pYES2 CT or pYES3 CT the presence of the 2u origin allows the plasmid to be episomally maintained and replicated at high copy number generally 10 40 copies per cell Continued on next page Cloning into pYES2 CT pYES3 CT or pYC2 CT Continued CEN6 ARSH4 Sequence Cloning Considerations The pYC2 CT vector contains the CEN6 ARSH4 sequence Sikorski and Hieter 1989 for maintenance and replication in yeast The CEN6 ARSH4 sequence is a 518 bp hybrid DNA fragment that contains a yeast centromere sequence CEN and an autonomously replicating sequence ARS Sikorski and Hieter 1989 The CEN6 sequence is derived from the CEN6 locus of yeast chromosome 6 Panzeri and Philippsen 1982 while the ARSH4 sequence is derived from the yeast histone H4 associated ARS Bouton and Smith 1986 When placed in a heterologous expression plasmid i e pYC2 CT the presence of the CEN6 ARSH4 sequence allows non integrative centromeric maintenance and low copy number replication of the plasmid generally 1 2 copies per cell pYES and pYC vectors do not contain an ATG initiation codon for pro
30. ontaining 5 mL YPD medium with the INVSc1 yeast stab Incubate the cells at 30 C with shaking overnight or until the culture is turbid Add 1 mL sterile 80 glycerol and mix thoroughly Dispense the stock into cryovials and freeze at 80 C G Gp A Revive the yeast by transferring a small portion of the frozen sample onto an YPD agar plate For research use only Not intended for any animal or human therapeutic or diagnostic use Introduction Product Overview Description of the System pYES2 CT pYES3 CT and pYC2 CT are 6 0 kb 5 9 kb and 4 6 kb vectors respectively designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae Features of the vectors allow purification and detection of expressed proteins see pages 13 20 for more information The vectors contain the following elements Yeast GAL1 promoter for high level inducible protein expression in yeast by galactose and repression by glucose Giniger et al 1985 West et al 1984 see page 9 for more information Multiple cloning site MCS with 8 or 9 unique sites plus two BstX I sites to facilitate in frame cloning with the C terminal peptide see page 7 for more information C terminal peptide encoding the V5 epitope and a polyhistidine 6xHis tag for detection and purification of your recombinant fusion protein 2u origin for episomal maintenance and high copy replication pYES2 CT and pYES3 CT or CEN6 ARSH4 sequence f
31. or non integrative centromeric maintenance and low copy replication pYC2 CT URA3 or TRP1 auxotrophic marker for selection of yeast transformants see below Ampicillin resistance gene for selection in E coli The table below summarizes the specific elements found in each vector Vector MCS Auxotrophic Marker Origin pYES2 CT 9 unique sites plus URA3 2u two BstX I sites pYES3 CT 8 unique sites plus TRP1 2u two BstX I sites pYC2 CT 9 unique sites plus URA3 CEN6 ARSH4 two BstX I sites Continued on next page Product Overview Continued Experimental The table below outlines the major steps required to clone and express your gene Outline of interest in pYES2 CT pYES3 CT or pYC2 CT Step Action 1 Consult the multiple cloning site described on page 7 to determine a strategy to clone your gene in frame with the C terminal peptide 2 Ligate your insert into the appropriate vector and transform into E coli Select transformants on LB plates containing 50 to 100 ug mL ampicillin 3 Analyze your transformants for the presence of insert by restriction digestion 4 Select a transformant with the correct restriction pattern and sequence to confirm that your gene is cloned in frame with the C terminal peptide 5 Transform your construct into competent INVSc1 cells and select for the appropriate amino acid prototrophy 6 Test for expression of your recombinant protein by western blot an
32. per initiation of translation Be sure to design your insert to contain an ATG initiation sequence In addition to the initiation codon you may also include the yeast consensus sequence at the translation initiation site An example of the yeast consensus sequence is provided below where the ATG translation initiation codon is shown underlined A Y A A C A A C AATGTC T C Note that other sequences are also possible The prevalence of the TCT as the second codon is thought to contribute to stabilization under the N end rule Hamilton et al 1987 Although not as strong as the mammalian Kozak translation initiation sequence the yeast consensus sequence is thought to have a 2 3 fold effect on the efficiency of translation initiation To express your gene as a recombinant fusion protein you must clone your gene in frame with the C terminal peptide containing the V5 epitope and the polyhistidine 6xHis tag See the diagram on the next page to develop a cloning strategy Note that pYES2 CT pYES3 CT and pYC2 CT possess the same multiple cloning site If you wish to express your protein WITHOUT the C terminal peptide be sure to include a stop codon Continued on next page Cloning into pYES2 CT pYES3 CT or pYC2 CT Continued Multiple Cloning Below is a diagram of the multiple cloning site for pYES2 CT pYES3 CT and Site of pYES2 CT pYC2 CT Features of the GAL1 promoter are marked as previously described pYES3 CT and Ginige
33. promoter Transferring cells from glucose to galactose containing medium causes the GAL1 promoter to become de repressed and allows transcription to be induced Alternatively cells may be maintained in medium containing raffinose as a carbon source The presence of raffinose does not repress or induce transcription from the GAL1 promoter Addition of galactose to the medium induces transcription from the GAL1 promoter even in the presence of raffinose Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose when compared to those maintained in glucose You may choose to grow cells containing your pYES2 CT pYES3 CT or pYC2 CT construct in glucose or raffinose depending on how quickly you want to obtain your expressed protein after induction with galactose and on the toxicity of the expressed protein For more information about expression in yeast refer to the Guide to Yeast Genetics and Molecular Biology Guthrie and Fink 1991 For a protocol to induce expression of your fusion protein with galactose proceed to Time Course of Protein Induction by Galactose on the next page Continued on next page Expression of Recombinant Protein Continued Time Course of To induce expression of your protein of interest from the GAL1 promoter Protein Induction galactose is added to the medium For cells that have been maintained in glucose by Galactose recombinant fusion protein can be detected in as little as 4
34. r et al 1985 Johnston and Davis 1984 Yocum et al 1984 Restriction pYC2 CT sites are labeled to indicate the cleavage site The multiple cloning site has been confirmed by sequencing and functional testing The vector sequences of pYES2 CT pYES3 CT and pYC2 CT are available for downloading from www invitrogen com or from Technical Support see page 29 For maps and a description of the features of pYES2 CT pYES3 CT and pYC2 CT refer to pages 13 20 GAL1 promoter TATA box 300 TTAACAGATA TATAAATGCA AAAACTGCAT AACCACTTTA ACTAATACTT TCAACATTTT rm start of transcription m 360 CGGTTTGTAT TACTTCTTAT TCAAATGTAA TAAAAGTATC AACAAAAAAT TGTTAATATA GAL1 forward priming site 3 end of GALT promoter PEER 420 CCTCTATACT TTAACGTCAA GGAGAAAAAA CCCCGGATCG GACTACTAGC AGCTGTAATA T7 promoter priming site a Aspi 1e a sarl Pampi 1 480 CGACTCACTA TAGGGAATAT TAAGCTTGGT ACCGAGCTCG GATCCACTAG TAACGGCCGC BstX EcoR an If Norl Xho Xba I l l 540 CAGTGTGCTG GAATTCTGCA GATATCCAGC ACAGTGGCGG CCGCTCGAGT CTAGAGGGCC V5 epitope I 1 600 CTTCGAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Polyhistidine region Pme I me AAA l 649 CGT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACCC GCTGATCCTA Arg Thr Gly His His His His His His CYC1 reverse priming site Me ve ne Se aval 699 GAGGGCCGCA TCATGTAATT AGTTATGTCA CGCTTACATT CACGCCCTCC CCCCACATCC Please
35. ration of 1X and boil the sample for 5 minutes 9 Load 20 pg of lysate onto an SDS PAGE gel and electrophorese Use the appropriate percentage of acrylamide to resolve your recombinant protein Continued on next page 11 Expression of Recombinant Protein Continued Note Scale up of Expression for Purification Note Purification 12 The C terminal peptide containing the V5 epitope and the polyhistidine 6xHis tag will add approximately 5 kDa to the size of your protein Once you have determined the optimal induction time necessary to obtain maximal protein expression you may increase the protein yield by scaling up the procedure described on page 10 If you plan to use ProBond resin to purify your recombinant fusion protein see the Note below To prepare cell lysates from culture volumes over 1 liter we recommend that you use a bead beater Biospec Products Bartlesville OK to lyse the cells Refer to Current Protocols in Molecular Biology Ausubel et al 1994 for a suitable protocol to lyse cells with a bead beater If you are using breaking buffer see previous page for purification of your recombinant protein on ProBond do not include EDTA in this buffer as it will TM interfere with the binding of proteins on ProBond For help with purification of your recombinant fusion protein refer to the ProBond Purification System manual If you are using another type of resin refer to t
36. s Schiestl R H and Gietz R D 1989 High Efficiency Transformation of Intact Cells Using Single Stranded Nucleic Acids as a Carrier Curr Genet 16 339 346 Sikorski R S and Hieter P 1989 A System of Shuttle Vectors and Yeast Host Strains Designed for Efficient Manipulation of DNA in Saccharomyces cerevisiae Genetics 122 19 27 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Tschumper G and Carbon J 1980 Sequence of a Yeast DNA Fragment Containing a Chromosomal Replicator and the TRP1 Gene Gene 10 157 166 Continued on next page 31 References Continued West R W J Yocum R R and Ptashne M 1984 Saccharomyces cerevisiae GAL1 GAL10 Divergent Promoter Region Location and Function of the Upstream Activator Sequence UASG Mol Cell Biol 4 2467 2478 Yocum R R Hanley S R West J and Ptashne M 1984 Use of lacZ Fusions to Delimit Regulatory Elements of the Inducible Divergent GAL1 GAL10 Promoter in Saccharomyces cerevisiae Mol Cell Biol 4 1985 1998 2009 Life Technologies Corporation All rights reserved 32 Notes 33 Notes 34 invitrogen Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F
37. s page as a positive control for expression and a sample with no DNA as a negative control for transformation Select for transformants on SC minimal media lacking the appropriate amino acids as described below Transformants should exhibit the appropriate amino acid prototrophy see table below See the Appendix page 22 for a recipe to prepare SC minimal media Vector Selection Medium Expected Growth Phenotype pYES2 CT SC U Ura pYES3 CT SC W Trp pYC2 CT SC U Ura Once you have identified a transformant be sure to purify the colony and make a glycerol stock for long term storage Maintain yeast cells containing your pYES2 CT pYES3 CT or pYC2 CT construct in the appropriate selective medium SC U or SC W containing 2 glucose or 2 raffinose see the next page See the Appendix page 22 for a recipe for SC minimal medium Note The growth rate of yeast strains varies with the carbon source Yeast strains typically exhibit the fastest growth in medium containing glucose Expression of Recombinant Protein GAL1 Promoter In typical S cerevisiae laboratory strains i e INVSc1 transcription from the GAL1 promoter is repressed in the presence of glucose West et al 1984 Transcription may be induced by removing glucose and adding galactose as a carbon source Giniger et al 1985 Maintaining cells in glucose gives the most complete repression and the lowest basal transcription of the GAL1
38. solution 10 fold with sterile deionized water 100 mM Lithium Acetate pH 7 5 5 mM Tris HCI pH 7 5 0 5 mM EDTA 1 For 100 mL mix together 10 mL of 10X LiAc and 5 mL of 10X TE 2 Add deionized water to 100 mL 3 Filter sterilize and store at room temperature 100 mM Lithium Acetate pH 7 5 40 PEG 3350 10 mM Tris HCl pH 7 5 1 mM EDTA 1 Prepare solution immediately prior to use For 100 mL mix together 10 mL of 10X LiAc 10 mL of 10X TE and 40 g of PEG 3350 2 Add deionized water to 100 mL and dissolve the PEG You may have to heat the solution to fully dissolve the PEG 3 Autoclave at 121 C 15 psi for 20 minutes Store at room temperature Small Scale Yeast Transformation Materials Needed YPD liquid medium 1X TE see Recipe page 24 1X LiAc 0 5X TE see Recipe page 24 Denatured salmon sperm DNA see recipe on the next page pYES2 CT pYES3 CT or pYC2 CT vector construct or other plasmid DNA to be transformed 1X LiAc 40 PEG 3350 1X TE See Recipe page 24 DMSO Selective plates Protocol 1 7 8 9 Inoculate 10 mL of YPD medium with a colony of INVSc1 and shake overnight at 30 C Determine the OD o of your overnight culture Dilute culture to an ODeo of 0 4 in 50 mL of YPD medium and grow an additional 2 4 hours Pellet the cells at 1 500 x g and resuspend the pellet in 40 mL 1X TE Pellet the cells at 1 500 x g and resuspend the pellet in 2 mL of 1X LiAc 0 5X
39. the control of the GAL1 promoter Successful transformation and galactose induction will result in B galactosidase expression that can be easily assayed see next page Continued on next page Yeast Transformation Continued Assay for galactosidase Activity Reagents for Yeast Transformation Yeast Transformation Maintaining Transformants You may assay for B galactosidase expression by activity assay using cell free lysates Miller 1972 Invitrogen offers the B Gal Assay Kit for fast and easy detection of B galactosidase expression see page 27 for ordering Many protocols are suitable for the preparation of competent INVSc1 yeast cells The S c EasyComp Kit provides a quick and easy method for preparing competent yeast cells that can be used immediately or stored frozen for future use see page 27 for ordering Transformation efficiency is guaranteed at gt 10 transformants per ug DNA A small scale yeast transformation protocol is included in the Appendix see page 25 for your convenience Alternatively there are published references for other small scale transformation methods Gietz et al 1992 Gietz et al 1995 Hill et al 1991 Schiestl and Gietz 1989 Use one of the methods described above or one of your own choosing to transform your pYES2 CT pYES3 CT or pYC2 CT plasmid construct into competent INVSc1 We recommend that you include the appropriate control vector see the previou
40. the sense orientation and sequencing through the insert Multiple cloning site with 9 unique sites and two BstX I sites Allows insertion of your gene and facilitates cloning in frame with the V5 epitope and the polyhistidine tag V5 epitope Permits detection of the fusion protein with the Anti V5 Antibody or the Anti V5 HRP Antibody Southern et al 1991 C terminal polyhistidine 6xHis tag Permits purification of your fusion protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody and the Anti His C term HRP Antibody Lindner et al 1997 CYC1 transcription termination signal Permits efficient termination and stabilization of mRNA CYC1 reverse priming site Allows sequencing through the insert pUC origin Allows maintenance and high copy replication in E coli Ampicillin resistance gene Allows selection of transformants in E coli URA3 gene Permits selection of yeast transformants in uracil deficient medium 2p origin Permits episomal maintenance and high copy replication in yeast fl origin Allows rescue of single stranded DNA pYES2 CT lacZ Description pYES2 CT lacZ is a 8971 bp control vector containing the gene for B galactosidase This vector was constructed by ligating a 3 1 kb fragment containing the lacZ gene into the BamH I Not I site of pYES2
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