GeneStorm clones - Thermo Fisher Scientific
Contents
1. Centrifuge the cells at 1500 rpm for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store at 70 C until needed If you are using ProBond resin refer to the ProBond Purification manual for details about sample preparation for chromatography This manual is available for downloading from our website at www invitrogen com If you are using other metal chelating resin refer to the manufacturer s instruction for recommendations on sample preparation Appendix Map of pcDNA 3 1 GS Map of The following map shows the elements of pcDNA 3 1 GS The full sequence pcDNA 3 1 GS of this vector excluding the insert is available on our website at www invitrogen com or by contacting Technical Support see page 9 Bst Il ORF Insert 902 AAGCTCGCCCTIICACCATG AAG GGC GAG CTT CGA GGT CAC TTCGAGCGGGAAGTGGTAC PTC CCG CTC GAA GCT CCA GTG Lys Gly Glu Leu Arg Gly His gt E 13 a XL Qa V5 epitope Note The underlined sequence CACC is not found in all clones Comments for pcDNA 3 1 GS no insert 4020 nucleotides CMV promoter bases 209 863 T7 promoter priming site bases 863 882 hORF cloning site between base 913 and 914 V5 epitope bases 944 985 Polyhistidine tag bases 995 1012 BGH Reverse priming site bases 1035 1052 BGH polyadenylation signal bases 1034 1248 f1 origin bases 1311 1724 SV40 promoter and origin bases 1789 2114 EM 7 promoter2. 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 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2000 2008 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 11 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
3. outline to express your GeneStorm Clone from pcDNA 3 1 GS Isolate plasmid DNA for transfection into the cell line of choice Be Transfect into the cell line of choice EN Prepare cell lysates for western blot analysis 3 4 Test for expression of the gene by western blot analysis or 4 functional assay 5 Purify the fusion protein using a metal chelating resin such as ProBond Methods Isolating Plasmid DNA Introduction Important Preparing Glycerol Master Stocks Growing E coli Cultures Plasmid Preparation This section describes how to isolate plasmid DNA for transfection into the cell line of choice To prepare plasmid DNA you need to prepare Low Salt LB medium with Zeocin For your convenience Low Salt LB medium containing 25 ug ml Zeocin is available as premixed pre sterilized E coli growth medium imMedia which contains everything you need in a convenient pouch see page vi TM For Zeocin to be active the salt concentration of the medium must remain low lt 90 mM and the pH must be 7 5 For selection in E coli it is imperative that you prepare LB broth and plates using the following recipe Failure to use Low Salt LB medium will result in non selection due to inactivation of the drug Refer to the Appendix page 8 for the recipe for Low Salt LB medium We recommend you prepare a set of master stocks prior to using your GeneStorm clone To prepare 5 10 glycerol maste
4. 620 21 Lipofectamine 2000 Reagent 0 75 ml 11668 027 15ml 11668 019 Detection and Antibodies and ProBond resin are available from Invitrogen See table below for Purification of ordering information Fusion Protein Item Amount Cat no Anti V5 HRP Antibody 50 ul R961 25 Anti V5 Antibody 50 ul R960 25 WesternBreeze Chemiluminescent 1 kit WB7104 Immunodetection Kit ProBond Purification System 6 purifications K850 01 ProBond Nickel Chelating Resin 50 ml R801 01 Precharged resin as a 50 slurry in 20 ethanol vi Overview Introduction Note Experimental Outline Introduction GeneStorm Expression Ready Clones are expressed from the vector pcDNA 3 1 GS This vector utilizes the strong immediate early cytomegalovirus CMV promoter for high level constitutive expression in mammalian cell lines Each GeneStorm Clone is fused to a C terminal peptide encoding the V5 epitope for detection with the Anti V5 HRP Antibody and a 6xHis tag for purification on metal chelating resin ie ProBond The vector also encodes the Zeocin resistance gene for selection in E coli and for the creation of stable mammalian cell lines For a map of peDNA 3 1 GS see page 7 For more information on GeneStorm Expression Ready Clones visit the website at www invitrogen com clones The GeneStorm Clones are not guaranteed to exactly match GenBank sequences and may differ by one or more bases Use the following
5. ation Transfection Introduction Methods of Transfection NA 7 NANO 7 e A 2 Detecting Fusion Proteins Cell Lysis Once you have isolated plasmid DNA you are ready to transfect your cell line of choice Supercoiled pcDNA 3 1 GS is included as a negative expression control Once you have confirmed expression of the gene by transient expression you may create stable cell lines using Zeocin as a selection agent For more information refer to the Zeocin manual available on our website at www invitrogen com or contact Technical Support see page 9 Methods for transfection include calcium phosphate Chen and Okayama 1987 Wigler et al 1977 lipid mediated Felgner et al 1989 Felgner and Ringold 1989 and electroporation Chu et al 1987 Shigekawa and Dower 1988 Consult published literature or the supplier of your cell line for the recommended method of transfection and transfection reagent to use For high efficiency transfection in a broad range of mammalian cell lines we recommend using Lipofectamine 2000 Reagent available from Invitrogen see page vi for ordering information For more information on a large selection of transfection reagents available from Invitrogen refer to our website at www invitrogen com or contact Technical Support see page 9 Supercoiled pcDNA 3 1 GS is supplied as a negative control for expression in mammalian cells We recommend that you include the negative co
6. bases 2130 2196 Zeocin resistance gene bases 2197 2571 SV40 polyadenylation bases 2580 2782 pUC origin bases 3214 3887 opposite strand Recipes Low Salt LB Medium with Zeocin Cell Lysis Buffer 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 ml Adjust pH to 7 5 with 5 M NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving 2 Autoclave on liquid cycle at 15 Ibs sq in and 121 C for 20 minutes Thaw Zeocin on ice and vortex before removing an aliquot Allow the medium to cool to at least 55 C before adding the Zeocin to 25 ug ml final concentration 5 Store plates at 4 C in the dark Plates containing Zeocin are stable for 1 2 weeks 50 mM Tris pH 7 8 150 mM NaCl 1 Nonidet P 40 1 This solution can be prepared from the following common stock solutions For 100 ml combine 1M Tris base 5 ml 5M NaCl 3 ml Nonidet P 40 1ml 2 Bring the volume up to 90 ml with deionized water and adjust the pH to 7 8 with HCl 3 Bring the volume up to 100 ml Store at room temperature Note Protease inhibitors may be added fresh at the following concentrations 1mM PMSF 1 pg ml pepstatin 1 ug ml leupeptin Technical Sup Web Resources port Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formu
7. eyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without limitation quality control and commercial services such as re porting the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 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 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Molec Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells Bio Techniques 6 742 751 Southern J A Young
8. hantability or fitness for a particular purpose Purchaser Notification Product Use by European Customers Limited Use Label License No 22 Vectors and Clones Contain ing Sequences Coding for Histidine Hexamer Limited Use Label License No 87 GeneStorm Clones Limited Use Label License No 358 Research Use Only 10 These cells are genetically modified and contain the pUC derived plasmid pcDNA3 1 GS As a condition of sale this product must be used only according to applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms 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 Life Technologies disclaims any warranty that the DNA inserted into the clones or the manufacture use or sale of those inserts or their sequences are free from third party intellectual property claims The purchase of this product conveys to the purchaser the limited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its components is conv
9. invitrogen GeneStorm Expression Ready Clones Genes Expressed from pcDNA 3 1 GS Cat no H K1000 IMPORTANT Version K Sui slik Beginning in 2009 11 November 2010 all Invitrogen clone 25 0221 manuals will only be available online at www invitrogen com 11 Table of Contents KIEBONTENIS nes nen v Accessory Producte manner vi OVEIVIEW aneignen 1 Isolating Plasma DNA cocos ee ei 2 LP et ee 3 Western Bl Analysis 2 2 2er 4 A A obec inde Ee aia dee enschede 6 Map of PENAS raf GS a ean ae eR ae 7 OCDE cecocg cuss tedattes Cada reen relia A A ia 8 Technical SUPPOF oe ei ee ee ee ee 9 Purchaser Notification oooooocccccnnnnnccccnnocccccoconononcnnannnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnns 10 Helerenees O 12 111 iv Kit Contents Shipping Storage GeneStorm Expression Ready Clones Kit Contents Each GeneStorm Expression Ready Human Clone is shipped on dry ice Upon receipt e Store the GeneStorm glycerol stock at 80 C e Store the rest of the kit at 4 C GeneStorm Expression Ready Clones Cat no H K1000 contain your gene of interest from the human genome All clones are supplied transformed into GeneHogs Electrocomp E coli All GeneStorm Clones expressed from pcDNA 3 1 GS are supplied with Anti V5 HRP Antibody and supercoiled pcDNA 3 1 GS The table below describes each component included in the kit Item Composition Amount Storage Ge
10. lations 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 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 3 5730 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 MSDSs Material Safety Data Sheets are available on our website at www invitrogen com msds The Certificate of Analysis CofA provides detailed quality control information for each product The CofA for each product is available on our website at www invitrogen com support and is searchable by product lot number which is printed on each box Limited Warranty Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied wi
11. neStorm Clone Supercoiled vector containing the gene of 1 5 ml 80 C interest transformed into GeneHogs Electrocomp E coli in LB media containing 10 glycerol and 25 ug ml Zeocin pcDNA 3 1 GS Supercoiled vector lyophilized in TE pH 8 0 50 ng 4 C Anti V5 HRP Antibody 1 mg ml in filter sterilized PBS see label on 25 ul 4 C tube for actual concentration 12 westerns Genotype of GeneHogs Anti V5 HRP Antibody F mcrA A mrr hsdRMS mcrBC o80lacZ M15 Alac 74 recAl araD139 A ara leu 7697 galU galK rpsL Str endA1 nupG fhuA 182 confers phage T1 resistance This antibody detects a 14 amino acid epitope GKPIPNPLLGLDST derived from the P and V proteins of the paramyxovirus SV5 Southern et al 1991 Accessory Products Additional Additional products are available from Invitrogen to help you characterize your Products GeneStorm Expression Ready Clone Item Amount Cat no GeneHogs Electrocomp E coli 5 x 100 ul C800 05 One Shot GeneHogs Electrocomp E coli 11 x 50 ul C8080 10 21 x 50 ul C8080 03 ChargeSwitch Pro Plasmid Miniprep Kit 10 preps CS30010 50 preps CS30050 250 preps CS30250 PureLink HiPure Plasmid MiniPrep Kit 25 preps K2100 02 PureLink HiPure Plasmid MidiPrep Kit 25 preps K2100 04 Zeocin lg R250 01 58 R250 05 imMedia Zeo Liquid 20 pouches Q620 20 imMedia Zeo Agar 20 pouches Q
12. ntrol in your transfection experiments The Anti V5 HRP Antibody is included in the kit to detect expression of the gene To detect the gene fusion protein by western blot you will need to prepare a cell lysate from transfected cells see next page We recommend that you perform a time course to optimize expression of the fusion protein e g 24 48 72 hours etc after transfection To lyse cells for western blot analysis 1 Wash cell monolayers 10 cells per 60 mm plate 80 90 confluent once with phosphate buffered saline PBS 2 Scrape cells into 1 ml PBS and pellet the cells at 1500 x g for 5 minutes 3 Resuspend in 50 ul Cell Lysis Buffer see recipe on page 8 Other lysis buffers may be suitable 4 Incubate cell suspension at 37 C for 10 minutes to completely lyse the cells You may also incubate at room temperature or on ice if you are concerned about protein degradation 5 Centrifuge the cell lysate at 10 000 x g for 10 minutes at room temperature to pellet nuclei and transfer the supernatant to a fresh tube 6 Assay the lysate for protein concentration Note Do not use protein assays utilizing Coomassie Blue or other dyes NP 40 interferes with the binding of the dye with the protein Proceed to Western Blot Analysis next page Western Blot Analysis Recommended Antibody Dilution Suggested Solutions Immunoblotting Protocol For western blots dilute the Anti V5 HRP Antibody 1 5000 into Pho
13. r stocks for long term storage 1 Streak a small portion of the glycerol stock you received out on a Low Salt LB plate containing 25 ug ml Zeocin see page 8 2 Incubate the plate at 37 C overnight 3 Isolate a single colony and inoculate into 5 10 ml of Low Salt LB containing TM 25 ug ml Zeocin 4 Grow the culture to stationary phase OD 1 2 5 Mix 0 80 ml of culture with 0 20 ml of sterile glycerol and transfer to a cryovial Store at 80 C Use one master stock to create working stocks for regular use We also recommend that you isolate and store a stock of plasmid DNA at 20 C To isolate plasmid DNA you need to grow a culture of GeneHogs containing your GeneStorm clone of interest Use Low Salt LB medium containing 25 ug ml Zeocin see recipe on page 8 to select single colonies or to grow a culture Use a culture volume appropriate for the amount of plasmid needed using your plasmid isolation method of choice Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipids decreasing transfection efficiency We recommend isolating plasmid DNA using a resin based method such as the ChargeSwitch Pro Plasmid Miniprep Kit up to 20 ug DNA PureLink HiPure Plasmid Miniprep Kit up to 30 ug DNA or PureLink HiPure Plasmid Midiprep Kit 100 350 ug DNA See page vi for ordering inform
14. ression of the GeneStorm Clone you may create stable cell lines or purify the protein To select stable cell lines refer to the Zeocin manual available on our website at www invitrogen com To purify your protein see the next page Purification Introduction Cell Preparation Lysis of Cells To obtain the highest yields possible we recommend that you purify your protein from a stable cell line ProBond is a metal chelating resin that can be used to purify recombinant proteins containing a polyhistidine 6xHis tag Ordering information on ProBond is on page vi If you are using other metal chelating resin follow the manufacturer s recommended procedure to purify your protein You will need 5 x 10 to 1 x 107 transfected cells for purification of your protein on a 2 ml ProBond column or other metal chelating column Refer to the manufacturer s instructions before attempting to purify your fusion protein To prepare cells for lysis follow the protocol below 1 Seed cells in either five T 75 flasks or 2 to 3 T 175 flasks 2 Grow the cells in selective medium until they are 80 90 confluent 3 Harvest the cells by treating with trypsin EDTA for 2 to 5 minutes or by scraping the cells in PBS 4 Inactivate the trypsin by diluting with fresh medium if necessary and transfer the cells to a sterile microcentrifuge tube 5 Centrifuge the cells at 1500 rpm for 5 minutes Resuspend the cell pellet in PBS 6
15. sphate Buffered Saline PBS containing 0 05 Tween 20 and 5 nonfat dry milk PBSTM If you use a different buffer for washing and blocking your blots then dilute as described above with that buffer You may use other blocking agents such as bovine serum albumin BSA or gelatin We use chemiluminescence to detect binding of the Anti V5 HRP Antibody to the recombinant protein Other detection methods can be used to detect your protein The following materials and solutions are needed for immunoblotting e Phosphate Buffered Saline 137 mM NaCl 2 7 mM KCI 10 mM Na HPO 1 8 mM KH2PO4 e Phosphate Buffered Saline Tween 20 PBST PBS plus 0 05 Tween 20 v v e Blocking buffer PBST 5 nonfat dry milk w v Use the protocol below to prepare your lysate sample for SDS PAGE and immunoblotting 1 Add SDS sample buffer to a final concentration of 1X to the lysate and heat the sample for 5 minutes at 70 C 2 Load 10 20 pg of protein onto an SDS PAGE and electrophorese Use the appropriate percentage of acrylamide to resolve the protein product Proceed to western transfer Note We use Novex 12 Tris Glycine polyacrylamide gels 3 Transfer proteins to nitrocellulose membrane electrophoretically We use 25 mM Tris pH 8 3 192 mM glycine 20 v v methanol as a transfer buffer 4 Run at 100V 150 mA 100V 240 mA at the finish for 1 hour Be sure to have a cooling system in place and operational with these electrophoretic se
16. th 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 the 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 please report it to our Technical Support Representatives Invitrogen 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 merc
17. ttings You may also transfer overnight at 30V 40 mA 30V 90 mA at the finish 5 Remove the nitrocellulose membrane and incubate it in 10 ml blocking buffer Gently agitate using a rocker platform for 1 hour at room temperature 6 Wash the nitrocellulose membrane in 20 ml PBST 2X for 5 minutes each with gentle agitation 7 Transfer the membrane to a tray containing the Anti V5 HRP Antibody diluted 1 5000 in 10 ml blocking buffer 2 ul of Anti V5 Antibody diluted into 10 ml blocking buffer Incubate with gentle agitation for 1 2 hours Note Overnight incubation may be preferred since longer incubations may increase the sensitivity of detection Generally 1 hour incubation is sufficient for detection 8 Transfer membrane to a tray containing 20 ml PBST and wash for 2 x 5 minutes with gentle agitation Proceed to detection next page Continued on next page Western Blot Analysis Continued Detection Reaction Note The Next Step We use chemiluminescence to detect the fusion proteins The WesternBreeze Chemiluminescent Immunodetection Kit is available from Invitrogen Cat no WB7104 Follow the manufacturer s instructions Other detection methods are suitable The C terminal peptide containing the V5 epitope and the polyhistidine tag will add approximately 3 kDa to the size of your protein In addition posttranslational modification may cause the protein to migrate differently than expected Once you have confirmed exp
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