pYES2 - Thermo Fisher Scientific
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1. coli or any other recA endA E coli strain of your choice Select for transformants on LB plates containing 50 100 ug ml ampicillin Select 10 20 clones and analyze by restriction digest or sequencing for the presence and orientation of your insert The T7 Forward primer is available from Invitrogen see page v to allow sequencing from the T7 promoter priming site see diagram on page 3 for the location of the priming site Alternatively you may wish to design primers that flank your gene of interest to verify the orientation of your insert in pYES2 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 in case you lose the glycerol stock 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 4 Mix 0 85 ml of 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 TM TM We rec2. assumes 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 17 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 141 Expression of Polypeptides in Yeast The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may inc
3. deficient medium 2p origin Maintenance and high copy replication in yeast fl origin Rescue of single stranded DNA Technical Support Web Resources Visit the Invitrogen web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service 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 Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 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 Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 35730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty MSDSs Material Safety Data Sheets are available on our web site at www invitrogen com msds Product qualification is described
4. g in 90 ml of deionized water Bring volume up to 100 ml Filter sterilize 2 Prepare 100 ml of 1 M NaHPO 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 1 M NaH PO and 77 4 ml of 1 M Na2HPO Bring the volume up to 1 liter with deionized water 4 Filter sterilize and store at room temperature 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 Continued on next page 11 Recipes continued 1X TE 10X LiAc 1X LiAc 1X LiAc 0 5X TE 1X LiAc 40 PEG 3350 1X TE 10 mM Tris pH 7 5 1mM EDTA Dilute 10X TE 10 fold with sterile water 1M 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 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
5. into cryovials and freeze at 80 C SAE Revive the yeast by transferring a small portion of the frozen sample onto an YPD agar plate Accessory Products Additional Products Invitrogen offers a variety of products that are suitable for use with the pYes2 plasmid Ordering information is provided below For detailed instructions on how to use any of the accessory products refer to the manual provided with each product For more information refer to www invitrogen com or contact Technical Support page 17 Item Amount Cat no PureLink HiPure Plasmid Miniprep Kit 25 preps K2100 02 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 50 preps K2100 05 Ampicillin Sodium Salt irradiated 200 mg 11593 027 One Shot TOP10 Chemically Competent E coli 10 reactions C4040 10 20 reactions C4040 03 Subcloning Efficiency DH5a Chemically 40 reactions 18265 017 Competent E coli T7 Forward primer 2ug N560 02 S c EasyComp Kit 1kit K5050 01 UltraPure Salmon Sperm DNA Solution 5x1ml 15632 011 10 mg ml Overview Introduction Experimental Outline Introduction pYES2 is a 5 9 kb vector designed for inducible expression of recombinant proteins in Saccharomyces cerevisiae Features of the vector allow easy cloning of your gene of interest and selection of transformants by uracil prototrophy see pages 15 16 The vector contains the following el
6. 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 80 ml of 50 PEG 3350 2 Filter sterilize and store at room temperature Small Scale Yeast Transformation Introduction A small scale yeast transformation protocol for routine transformations is provided below Other protocols are suitable Materials Needed YPD liquid medium 1X TE see Recipe page 12 1X LiAc 0 5X TE see Recipe page 12 Denatured salmon sperm DNA see recipe on the next page pYES2 vector construct or other plasmid DNA to be transformed 1X LiAc 40 PEG 3350 1X TE See Recipe page 12 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 of your overnight culture Dilute culture to an ODsoo of 0 4 in 50 ml of YPD medium and grow an additional 2 4 hours Pellet the cells at 2500 rpm and resuspend the pellet in 40 ml 1X TE Pellet the cells at 2500 rpm and resuspend the pellet in 2 ml of 1X LiAc 0 5X 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
7. research use only Not intended for any animal or human therapeutic or diagnostic use 19 20 Notes invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
8. 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 Your insert must also contain a stop codon for proper termination of your mRNA Note that the Xba I site contains an internal stop codon TCTAGA Continued on next page Cloning into pYES2 continued Multiple Cloning Site of pYES2 Below is a diagram of the GAL1 promoter and the multiple cloning site for pYES2 Features of the GAL1 promoter are marked as per Giniger et al 1985 Johnston and Davis 1984 and Yocum et al 1984 Restriction sites are labeled to indicate the cleavage site Potential stop codons are underlined The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pYES2 is available for downloading from our website www invitrogen com or from Technic
9. 13 Ausubel et al 1994 for a suitable protocol to lyse cells with a bead beater Recipes SC Minimal Medium and Plates Induction Medium Note Appendix SC is synthetic minimal defined medium for yeast 0 67 yeast nitrogen base without amino acids 2 carbon source i e glucose or raffinose 0 01 adenine arginine cysteine leuine 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 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 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 5 Pour plates and allow to harden Invert the plates and store at 4 C Plates are stable for 6 months If you are making indu
10. 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 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nuc Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biol 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press 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 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 UASc 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 1998 2008 Invitrogen Corporation All rights reserved For
11. al Support see page 17 5 end of GAL1 promoter GAL4 binding site GAL4 binding site GAL4 binding site I LI ACGGATTAGA AGCCGCCGAG CGGGTGACAG CCCTCCGAAG GAAGACTCTC CTCCGTGCGT GAL4 binding site CTCGCGCCGC ACTGCTCCGA 61 CCTCGTCTTC ACCGGTCGCG TTCCTGAAAC GCAGATGTGC 121 ACAATAAAGA TTCTACAATA CTAGCTTTTA TGGTTATGAA GAGGAAAAAT TGGCAGTAAC 181 CTGGCCCCAC AAACCTTCAA ATGAACGAAT CAAATTAACA ACCATAGGAT GATAATGCGA 241 TTAGTTTTTT AGCCTTATTT CTGGGGTAAT TAATCAGCGA AGCGATGATT TTTGATCTAT TATA box 301 TAACAGATAT ATAAATGCAA AAACTGCATA ACCACTTTAA CTAATACTTT CAACATTTTC start of transcription 361 GGTTTGTATT ACTTCTTATT CAAATGTAAT AAAAGTATCA ACAAAAAATT GTTAATATAC 3 end of GAL1 promoter p on 421 CTCTATACTT TAACGTCAAG GAGAAAAAAC CCCGGATCGG ACTACTAGCA GCTGTAATAC T7 promoter priming site Hind Ill Kpnl Sac BamH ___ ll 481 GACTCACTAT AGGGAATATT AAGCTTGGTA CCGAGCTCGG ATCCACTAGT AACGGCCGCC BstX EcoRI BsaB BstX I Not ano l Sph Xba l l l 541 AGTGTGCTGG AATTCTGCAG ATATCCATCA CACTGGCGGC CGCTCGAGCA TGCATCTAGA 5 end of CYC1 transcription terminator 601 GGGCCGCATC ATGTAATTAG TTATGTCACG CTTACATTCA CGCCCTCCCC CCACATCCGC Note that there are two BstX I sites in the polylinker Continued on next page Cloning into pYES2 continued E coli Transformation Sequencing Inserts in pYES2 Preparing a Glycerol Stock Plasmid Preparation Transform your ligation mixtures into competent TOP10F E
12. ansform your pYES2 plasmid construct into competent INVSc1 We recommend that you include the pYES2 parental vector as a negative control to evaluate your results Select for transformants on SC U selective plates Transformants should exhibit uracil prototrophy Once you have identified a transformant be sure to purify the colony and make a glycerol stock for long term storage Maintain cells containing your pYES2 construct in SC U medium containing 2 raffinose or 2 glucose See the Appendix page 10 for a recipe for SC U medium Expression of Recombinant Protein Introduction GAL1 Promoter Once you have obtained a transformant containing your pYES2 construct you are ready to induce expression of your recombinant protein of interest This section provides information on how to induce and assay for expression of your protein of interest 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 GALI 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 contain
13. c research use only For profit 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 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 Binding 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 Hamilton R Watanabe C K and de Boer H A 1987 Compilation and comparison of the sequence context around the AUG startcodons in Saccharomyces cerevisiae mRNAs Nucleic Acids Res 15 3581 3593 Hill J Donald K A and Griffiths D E 1991 DMSO Enhanced
14. ction 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 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 Continued on next page Recipes continued YPD 0 1 M Sodium Phosphate pH 7 4 10X TE 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 has been autoclaved add 100 ml of 20 dextrose to the medium Materials needed Sodium phosphate monobasic NaH2PO4 H20 Sigma Aldrich 9638 Sodium phosphate dibasic NazHPO 7H20 Sigma Aldrich 59390 Protocol 1 Prepare 100 ml of 1 M NaH PO H 0 by dissolving 13 8
15. ements e 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 7 for more information e A versatile multiple cloning site for simplified cloning e CYC1 transcriptional terminator for efficient termination of mRNA e URA3 gene for selection of transformants in yeast host strains with a ura3 genotype e Ampicillin resistance gene for selection in E coli Use the following outline to clone and express your gene of interest in pYES2 1 Consult the multiple cloning site described on page 3 to design a strategy to clone your gene in pYES2 2 Ligate your insert into pYES2 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 use sequencing to confirm that your gene is cloned in the proper orientation 5 Transform your construct into competent INVSc1 cells and select for uracil prototrophy 6 Test for expression of your recombinant gene by western blot analysis or functional assay Methods Cloning into pYES2 Introduction General Molecular Biology Techniques E coli Strain Transformation Method Maintaining pYES2 Cloning Considerations A diagram is provided on the next page to help you ligate your ge
16. figure below summarizes the features of the pYES2 vector The vector sequence of pYES2 is available for downloading from our website www invitrogen com or from Technical Support see page 17 SnaB Bgl Comments for pYES2 5856 nucleotides Cla GAL1 promoter bases 1 451 T7 promoter priming site bases 475 494 Multiple cloning site bases 501 600 CYC1 transcription terminator bases 608 856 pUC origin bases 1038 1711 Ampicillin resistance gene bases 1856 2716 C URA3 gene bases 2734 3841 C 2 micron p origin bases 3845 5316 f1 origin bases 5384 5839 C C complementary strand Continued on next page 15 pYES2 Vector continued Features of pYES2 pYES2 5856 bp contains the following elements All features have been functionally tested Feature Benefit GALI promoter Permits inducible expression of genes cloned into pYES2 West et al 1984 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 plus two BstX I sites Allows insertion of your gene into pYES2 CYC1 transcription termination signal Permits efficient termination and stabilization of mRNA pUC origin Maintenance and high copy replication in E coli Ampicillin resistance gene Selection of transformants in E coli URA3 gene Selection of yeast transformants in uracil
17. h a 10 ml pipette to dissolve completely Incubate overnight at 4 C 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 You may verify the size of the DNA ona 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 pheno 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 and 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 pYES2 Vector Map of pYES2 The
18. iate a culture of INVSc1 from the stab provided with the kit streak a small amount from the stab ona YPD plate see Appendix for recipe page 11 and incubate at 30 C Once growth is established you may check the phenotype of the strain by streaking a single colony on a 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 Continued on next page Yeast transformation continued Reagents for Yeast Transformation Yeast Transformation 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 v for ordering information Transformation efficiency is guaranteed at gt 10 transformants per ug DNA A small scale yeast transformation protocol is included in the Appendix see page 13 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 tr
19. in the Certificate of Analysis CofA available on our website by product lot number at www invitrogen com support Invitrogen 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 Invitrogen warrants that all of its products will 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 Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen 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 Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Invitrogen
20. ing raffinose as a carbon source The presence of raffinose does not repress or induce transcription from the GALI 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 construct in glucose or raffinose depending on how quickly you want to obtain your expressed protein after induction with galactose For more information about expression in yeast refer to the Guide to Yeast Genetics and Molecular Biology Guthrie and Fink 1991 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 raffinose by Galactose recombinant protein can be detected in as little as 2 hours after galactose induction Recombinant protein can be detected in cells that have been cultured in glucose by 4 hours after galactose induction If you are assaying for expression of your recombinant protein for the first time we recommend that you perform a time course to optimize expression of your recombinant protein e g 0 2 4 6 8 10 hours after galactose induction A standard protocol is provided below to perform a ti
21. invitrogen pYES2 Cat no V825 20 Version K 5 December 2008 28 0053 Table of Contents Table Of Contento ici ae AAU dl ic end iii Important informa unica tad tardan bin iv NN O NR SS CPTIELPOVE NC E EEEa ania v IntrodU CHO sss sanaan aaea anara aaa aAa ais 1 ONL 814 17 AEREE EEE E E E E E NN NA 1 Cloning into pY ESZ aaaea e ai e RS EA lalola RESE 2 Yeast Transformation aus 2a pnp nRa e R a oa E EN Eaa EA aE EE 5 Expression of Recombinant Protein ccccccccscescssssesesesesesesesnsnsnesesescsescececeueseseseeceeeesesesesesesesnansneseseseseeeeeeeeenenes 7 A A 10 Reeipesan a NO NN OO 10 Small Scale Yeast Transformati n 2 22 2 2 82822 32222 es Egg nebenbsligtes 13 Preparing Denatured Salmon Sperm DNA oococoncncoonnnnnnonnnnnonnnnnnnnnnnnnnnnorarannnanonnnnananananonnn nono nana a R REE 14 PYES2 Veelor in lia iii 15 Technical Sup A NN 17 Purchaser Notifica 0 A A AA a a ten 18 References cia ia A A daba 19 A NN 20 Important Information Kit Contents Shipping Storage Genotype of TOP10F Preparing TOP10F Glycerol Stocks Genotype Phenotype of INVSc1 Preparing INVSc1 Glycerol Stocks The following reagents are supplied with the pYES2 vector 20 ug of pYes2 plasmid in TE buffer pH 8 0 40 ul at 0 5 ug nl TOP10F E coli cells 1 stab INVSc1 yeast host strain 1 stab TE Buffer 10 mM Tris HCl 1 mM EDTA pH 8 0 All reagents are shipped at room temperature Upon receipt store the
22. llets in 500 pl 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 ODeo0 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 concentration 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 Scale up 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 8 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 Unit 13
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24. me course experiment Other protocols are suitable 1 Inoculate a single colony of INVSc1 containing your pYES2 construct into 15 ml of SC U medium containing 2 raffinose or 2 glucose Grow overnight at 30 C with shaking 2 Determine the ODso of your overnight culture Calculate the amount of overnight culture necessary to obtain an ODeq of 0 4 in 50 ml of induction medium Example Assume that the OD of your overnight culture is 3 ODeo0 per ml Then the amount of overnight culture needed to inoculate a 50 ml culture to OD6ev0 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 4 C 4 Resuspend the cells in 1 2 ml of induction medium SC U medium containing 2 galactose and inoculate into 50 ml of induction medium See the Appendix page 10 for a recipe for induction medium Grow at 30 C with shaking 5 Harvest an aliquot of cells at 0 2 4 6 8 and 10 hours after addition of cells to the induction medium For each time point remove 5 ml of culture from the flask and determine the ODeo of each sample 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 a
25. ne of interest into pYES2 General considerations for cloning and transformation are discussed below For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 We recommend that you propagate the pYES2 vector in the TOP10F E coli strain A stab of TOP10F is provided for your convenience Other E coli strains that are recombination deficient recA and endonuclease deficient endA are also suitable for the growth of this vector To initiate a culture of TOP10F from the supplied stab streak a small amount of stock from the stab on an LB plate containing 10 pg ml tetracycline You may use any method of your choice for transformation Chemical transformation is the most convenient for most researchers Electroporation is the most efficient and the method of choice for large plasmids To propagate and maintain the pYES2 vector use 10 ng of the vector to transform a recA endA E coli strain like TOP10F DH5a JM109 or equivalent Select transformants on LB plates containing 50 to 100 pg ml ampicillin Be sure to prepare a glycerol stock of the plasmid for long term storage see page 4 for a protocol pYES2 vector does not contain an ATG initiation codon for proper initiation of
26. ommend the PureLink HiPure Plasmid Miniprep Kit or the PureLink HiPure Plasmid Midiprep Kit for preparing plasmid DNA for routine yeast transformations see page v for ordering information Refer to our website at www invitrogen com or contact Technical Support for more information on a large selection of plasmid purification columns Yeast Transformation Introduction Basic Yeast Molecular Biology Genotype Phenotype of INVSc1 Initiating INVSc1 Culture In this section you will use a small scale yeast transformation protocol to transform your construct into the INVSc1 yeast host strain included with the vector The user should be familiar 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 MATa his3A1 leu2 trp1 289 ura3 52 MATa his3A1 leu2 trp1 289 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 and uracil A recipe for preparation of SC minimal medium is provided in the Appendix see page 10 To init
27. plasmid DNA at 20 C Store the TOP10F and INVSc1 stabs at 4 C The genotype of TOP10F is provided below F lacl4 Tn10 Tet mcrA A mrr hsdRMS mcrBC 80lacZAM15 AlacX74 endAl recA1 araD139 A ara leu 7697 galU galK nupG rpsL Str We recommend that you prepare a set of TOP10F E coli glycerol master stocks within two weeks of receiving the kit To prepare 5 10 glycerol master stocks for long term storage 1 Streak a small portion of the TOP10F cells that you have received as a stab on an LB plate Invert the plate and incubate at 37 C overnight Isolate a single colony and inoculate into 5 10 ml of LB medium Grow the culture to stationary phase OD 1 2 Mix 0 8 ml of culture with 0 2 ml of sterile glycerol and transfer to a cryovial DN OT RCO ah Store at 80 C Use one master stock to create working stocks for regular use The genotype and phenotype of the INVSc1 host strain are provided below Genotype MATa his3A1 leu2 trp1 289 ura3 52 MATa his3A1 leu2 trp1 289 ura3 52 Phenotype His Lew 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 containing 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
28. t 80 C until ready to use Proceed to the next section to prepare cell lysates to detect your recombinant protein see next page Continued on next page Expression of Recombinant Protein continued Detecting You may use any method of your choice to detect expression of your recombinant Recombinant protein from pYES2 If you wish to use western blot analysis to assay for protein Protein expression you will need to have an antibody to your protein of interest To detect the recombinant protein by western blot you 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 Unit 13 13 Ausubel et al 1994 for more information For large scale preparations culture volumes over 1 liter see Scale up below Materials Needed Breaking buffer 50 mM sodium phosphate pH 7 4 1 mM EDTA 5 glycerol 1 mM PMSF refer to Appendix page 11 for instructions to prepare the sodium phosphate stock buffer Glass beads acid washed 425 600 um size 30 40 U S sieve e g Sigma Aldrich Cat no G8772 Protocol 1 You may prepare cell lysates from either frozen cells or fresh cells Reminder You will need to know the ODgq0 of your cell sample s before beginning see Step 5 previous page 2 Resuspend fresh or frozen cell pe
29. yeast suspension from Step 5 Add 700 pl 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 pl 1X TE and plate on a selective plate Note To calculate the number of yeast cells assume that 1 ODso unit 2 0 x 10 yeast cells 13 Preparing Denatured Salmon Sperm DNA Introduction Materials Needed Protocol A convenient protocol to make denatured salmon sperm DNA Schiestl and Gietz 1989 is provided for your convenience You may also purchase denatured salmon sperm DNA from Sigma Aldrich Cat no D9156 Alternatively some researchers have found that using yeast transfer RNA Sigma Aldrich Cat no R9001 as a carrier results in a cleaner transformation although there are fewer total colonies 10 11 12 13 Salmon Sperm DNA Sigma Aldrich Cat no D1626 1X TE e Chloroform Sonicator e Low speed centrifuge 50 ml conical centrifuge tubes e 3M sodium acetate pH 6 0 TE saturated phenol e 95 ethanol 20 C 250 ml centrifuge bottle e 250 ml centrifuge bottle TE saturated phenol chloroform isoamyl alcohol 25 24 1 In a 250 ml flask dissolve 1 g salmon sperm DNA into 100 ml of TE 10 mg ml Pipette up and down wit
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