Manual Title - Thermo Fisher Scientific
Contents
1. GTACACCAAT TGACGTCAAT TTAGTTCATA GGCTGACCGC ACGCCAATAG TTGGCAGTAC AAATGGCCCG TACATCTACG GGGCGTGGAT GGGAGTTTGT GACTTTCCAA AATGTCGTAA TATA r TAACCCCGCC CCGTTGACGC pCEP Forward primer CGGTGGGAGG TCTATATAAG Kpn I Pvu Il Nhe I Hind III GTACCAGCTG CTAGCAAGCT CAGAGCTCGT TTAGTGAACC Nhe Not Xho TGCTAGCGGC CGCTCGAGGC EBV Reverse primer ATGATAAGAT ACATTGATGA GTTTGGACAA ACCACAACTA Continued on next page Cloning into pCEP4 Continued E coli Transformation Sequencing Your Construct Preparing a Glycerol Stock Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 and select transformants on LB plates containing 50 to 100 pg mL ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert Transformation Method 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 Several primers are available separately that you may use to sequence your construct These are marked in the multiple cloning site diagram on page 3 For ordering information see page 11 Alternatively you may design your own primer for sequencing Once you have identified the correct clone purify the colony and make a glycerol stock for long term storage2. You should 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 Grow the culture to mid log phase ODsoo 0 5 0 7 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C Transfection Introduction Plasmid Preparation Methods of Transfection Positive Control Assay for CAT Protein Once you have verified that your gene is cloned in the correct orientation and contains an initiation ATG and a stop codon you are ready to transfect your cell line of choice We recommend that you include the positive control vector and a mock transfection negative control to evaluate your results Plasmid DNA for transfection into eukaryotic cells must be clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HQ Mini Plasmid Purification Kit or PureLink HiPure Miniprep or Midiprep Kits available from Invitrogen see page 11 For established cell lines e g HeLa consult original references or the supplier of your cell line for the optimal method of transfection We recommend that you follow exactly the protoco
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4. invitrogen com or by contacting Technical Support page 12 vAygromycin Nru I Comments for pCEP4 CAT 10891 nucleotides CMV promoter bases 1 588 Chloramphenicol acetyl transferase CAT gene bases 675 1334 SV40 polyadenylation signal bases 1390 1631 OriP bases 2049 4024 EBNA 1 gene complementary strand bases 4325 6250 Ambpicillin resistance gene bases 6876 7736 pUC origin bases 7745 8520 TK promoter bases 8888 9050 Hygromycin resistance gene bases 9114 10124 TK polyadenylation signal bases 10136 10407 10 Accessory Products Introduction Primers The products listed below are designed for use with pBudCE4 1 For details visit www invitrogen com or contact Technical Support page 12 Assay Kit Item Quantity Catalog no One Shot TOP10 Chemically Competent Cells 21 x 50 pL C4040 03 One Shot TOP10 Electrocomp Cells 21 x 50 pL C4040 52 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 PureLink HQ Mini Plasmid Purification Kit 100 preps K2100 01 Lipofectamine 2000 Reagent 1 5 mL 11668 019 Hygromycin B 20 mL 10687 010 Fast Cat Chloramphenicol Acetyltransferase 1 kit F2900 For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details 11 Technical Support Web Resources Visit the Invitrogen website
5. this plasmid to permit extrachromosomal replication in human primate and canine cells pCEP4 also carries the hygromycin B resistance gene for stable selection in transfected cells pCEP4 CAT is provided as a positive control for the relative level of expression of recombinant proteins in a cell line of interest It expresses the chloramphenicol acetyl transferase CAT protein from the CMV enhancer promoter Like its parent vector pCEP4 pCEP4 CAT contains the hygromycin B resistance gene for selection Use the following outline to clone and express your gene of interest in pCEP4 1 Consult the multiple cloning site described on page 3 to design a strategy to clone your gene into pCEP4 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 Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation 5 Transfect your construct into the mammalian cell line of interest using your own method of choice Generate a stable cell line if desired 6 Test for expression of your recombinant gene by western blot analysis or functional assay Methods Cloning into pCEP4 General Molecular Biology Techniques E coli Strain Maintaining pCEP4 Important For help wi
6. art 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 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 consequent
7. asmid Encoded Hygromycin B Resistance The Sequence of Hygromycin B Phosphotransferase Gene and its Expression in E coli and S Cerevisine Gene 25 179 188 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 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 McKnight S L 1980 The Nucleotide Sequence and Transcript Map of the Herpes Simplex Virus Thymidine Kinase Gene Nucleic Acids Res 8 5949 5964 Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Palmer T D Hock R A Osborne W R A and Miller A D 1987 Efficient Retrovirus Mediated Transfer and Expression of a Human Adenosine Deaminase Gene in Diploid Skin Fibroblasts from an Adenosine Deficient Human Proc Natl Acad Sci U S A 84 1055 1059 Reisman D and Sugden B 1986 trans Activation of an Epstein Barr Viral Transcriptional Enhancer by the Epstein Barr Viral Nuclear Antigen 1 Mol Cell Biol 6 3838 3846 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloni
8. ial 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 References Andersson S Davis D L Dahlb ck H J rnvall H and Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 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 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 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 a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Gritz L and Davies J 1983 Pl
9. information and guidelines are provided in this section for your convenience Hygromycin B 527 5 MW is an aminocyclitol that inhibits protein synthesis by disrupting translocation and promoting mistranslation Hygromycin B phospho transferase detoxifies hygromycin B by phosphorylation e Hygromycin is light sensitive Store the liquid stock solution at 4 C protected from exposure to light e Hygromycin is toxic Do not ingest solutions containing the drug e Wear gloves a laboratory coat and safety glasses or goggles when handling hygromycin and hygromycin containing solutions To successfully generate a stable cell line expressing your gene of interest from pCEP4 you need to determine the minimum concentration of hygromycin B required to kill your untransfected host cell line Typically concentrations ranging from 10 to 400 pg mL hygromycin are sufficient to kill most untransfected mammalian cell lines We recommend that you test a range of concentrations see protocol below to ensure that you determine the minimum concentration necessary for your host cell line 1 Plate or split a confluent plate so the cells will be approximately 25 confluent Prepare a set of 7 plates Allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of hygromycin 0 10 25 50 100 200 400 ug mL hygromycin 3 Replenish the selective media every 3 4 days and observe the
10. invitrogen pCEP4 Catalog no V044 50 Rev date 28 December 2010 Manual part no 28 0038 MAN0000615 ii Table of Contents Kit Contents and Storage oasen roven ververst e nn na nn en NR ea iv Introduction ya ooo do 1 Product Overview A A in 1 Metodo 2 Cloning into pEEP4 comino abs See ae 2 MA gneis 5 Creating Stable Cell Lines anne Sesheh solids EE E EEE E PE E a E E ii 6 poa Lol AEP E EEE OS O A T 8 PEEPA Vect r nase A E E A A E 8 PCEPA CAT nd een a de dd 10 Accessory Products A Er SH Paola karene ENEE SEEE 11 Technical SUpport sisusse ioral eden Ea S Dat ui 12 References minnen dada 13 iii Kit Contents and Storage Shipping and Storage Kit Contents pCEP4 vectors are shipped on wet ice Upon receipt store vectors at 20 C All vectors are supplied as detailed below Store the vectors at 20 C Vector Composition Amount CEP4 40 uL of 0 5 ug uL vector in 10 mM Tris P HCI 1 mM EDTA pH 8 0 20 uL of 0 5 ug uL vector in 10 mM Tris PEER HCI 1 mM EDTA pH 8 0 20 ug 10 ug Introduction Product Overview pCEP4 Experimental Outline pCEP4 is an episomal mammalian expression vector that uses the cytomegalovirus CMV immediate early enhancer promoter for high level transcription of recombinant genes inserted into the multiple cloning site The Epstein Barr Virus replication origin oriP and nuclear antigen encoded by the EBNA 1 gene is carried by
11. l for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Ausubel et al 1994 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 Invitrogen offers the Lipofectamine 2000 Reagent for mammalian cell transfection see page 11 pCEP4 CAT is provided as a positive control vector for mammalian transfection and expression see page 10 and may be used to optimize transfection conditions for your cell line The gene encoding chloramphenicol acetyl transferase CAT is expressed in mammalian cells under the control of the CMV promoter A successful transfection will result in CAT expression that can be easily assayed see below You may assay for CAT expression using your method of choice Invitrogen offers a CAT assay kit for detection of the protein page 11 Creating Stable Cell Lines Introduction Hygromycin B Activity Determining Antibiotic Sensitivity pCEP4 contains the hygromycin resistance gene for selection of stable cell lines using hygromycin B We recommend that you test the sensitivity of your mammalian host cell to hygromycin B as natural resistance varies among cell lines General
12. lasmid Reisman and Sugden 1986 Yates et Transfectants al 1985 Transfected cells may lose the pCEP4 plasmid if they are not maintained under selection or are continuously cultured for long periods of time over six months To prevent loss of pCEP4 from transfected cells we recommend that you follow these guidelines when working with the cells e Always use early passage cells Grow and freeze multiple vials of transfected cells to ensure that you have an adequate supply of early passage cells e Always maintain cells in medium containing 50 ug mL hygromycin e Do not maintain cells in continuous culture for longer than 6 months Appendix pCEP4 Vector Map of pCEP4 The figure below summarizes the features of the pCEP4 vector The sequence for pCEP4 is available for downloading from our website www invitrogen com or from Technical Support see page 12 x Hind Ill Nhe Sfi BamH Nhe I Not eS S Comments for pCEP4 10186 nucleotides CMV promoter bases 1 588 Multiple cloning site bases 619 676 SV40 polyadenylation signal bases 685 926 OriP bases 1344 3319 EBNA 1 gene complementary strand bases 3620 5545 Ampicillin resistance gene bases 6171 7031 pUC origin bases 7040 7815 TK promoter bases 8183 8345 Hygromycin resistance gene bases 8409 9419 TK polyadenylation signal bases 9431 9702 Continued on next page pCEP4 Vector Continued functionally tested Feat
13. ng A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 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 Yates J L Warren N and Sugden B 1985 Stable Replication of Plasmids Derived from Epstein Barr Virus in Various Mammalian Cells Nature 313 812 815 2009 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 13 invitrogen Corporate Headquarters 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
14. ng neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG Your insert should also contain a stop codon for proper termination of your gene Continued on next page Cloning into pCEP4 Continued Multiple Cloning Site of pCEP4 61 121 181 241 301 361 421 481 541 601 661 Below is the multiple cloning site for pCEP4 Restriction sites are labeled to indicate the cleavage site The multiple cloning site has been confirmed by sequencing and functional testing The sequence of pCEP4 is available for downloading from our website www invitrogen com or from Technical Support see page 12 For a map and a description of the features refer to pages 8 9 GTTGACATTG GCCCATATAT CCAACGACCC GGACTTTCCA ATCAAGTGTA CCTGGCATTA ATTATTGACT AGTTATTAAT AGTAATCAAT p enhancer region 5 end GGAGTTCCGC GTTACATAAC TTACGGTAAA CCGCCCATTG TTGACGTCAA TCATATGCCA TGCCCAGTAC TATTAGTCAT CGCTATTACC AP1 Po AGCGGTTTGA CTCACGGGGA enhancer region 3 end TTTGGCACCA AAATCAACGG CAAT AAATGGGCGG TAGGCGTGTA Transcriptional start 7 GTCAGATCTC TAGAAGCTGG Sfi BamH CGGCAAGGCC GGATCCAGAC ACGTCAATAA TGGGTGGAGT AGTCCGCCCC ATGACCTTAC ATGGTGATGC TTTCCAAGTC TGACGTATGT ATTTACGGTA CTATTGACGT GGGACTTTCC GGTTTTGGCA TCCACCCCAT TACGGGGTCA TGGCCCGCCT TCCCATAGTA AACTGCCCAC CAATGACGGT TACTTGGCAG
15. percentage of surviving cells 4 Count the number of viable cells at regular intervals to determine the appropriate concentration of hygromycin that prevents growth within 2 3 weeks after addition of hygromycin Note Cells will divide once or twice in the presence of lethal doses of hygromycin so the effects of the drug may take several days to become apparent Complete inhibition of cell growth can take 2 3 weeks of growth in selective medium Continued on next page Creating Stable Cell Lines Continued Selecting Stable Once you have determined the appropriate hygromycin concentration to use for Integrants selection in your host cell line you can generate a stable cell line expressing your gene of interest 1 Transfect your mammalian host cell line with your pCEP4 construct using the desired protocol Remember to include a plate of untransfected cells as a negative control and the pCEP4 CAT plasmid as a positive control 24 hours after transfection wash the cells and add fresh medium to the cells 48 hours after transfection split the cells into fresh medium containing hygromycin at the pre determined concentration required for your cell line Split the cells such that they are no more than 25 confluent 4 Feed the cells with selective medium every 3 4 days until hygromycin resistant foci can be identified 5 Pick and expand colonies in 96 or 48 well plates Maintaining Stable pCEP4 is an episomally maintained p
16. th 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 Many E coli strains are suitable for the propagation of this vector We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA and endonuclease A deficient endA For your convenience TOP10 is available as chemically competent or electrocompetent cells from Invitrogen see page 11 To propagate and maintain pCEP4 use a small amount of the supplied 0 5 ug uL stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10 or equivalent Select transformants on LB plates containing 50 to 100 ug mL ampicillin Be sure to prepare a glycerol stock of your plasmid containing E coli strain for long term storage see page 4 Your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1990 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while havi
17. ures of pCEP4 pCEP4 10 186 bp contains the following elements All features have been Feature Benefit Human cytomegalovirus CMV immediate early Allows efficient high level expression of your recombinant protein Andersson promoter enhancer et al 1989 Boshart et al 1985 Nelson et al 1987 Multiple cloning site Allows insertion of your gene and facilitates cloning SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA EBV origin of replication oriP and nuclear antigen EBNA 1 High copy episomal replication in primate and canine cell lines Reisman and Sugden 1986 Yates et al 1985 Ampicillin resistance gene B lactamase Selection of vector in E coli pUC origin High copy number replication and growth in E coli Herpes Simplex Virus thymidine kinase TK promoter Allows efficient high level expression of the hygromycin resistance gene MeKnight 1980 Hygromycin resistance gene Selection of stable transfectants in mammalian cells Gritz and Davies 1983 Palmer et al 1987 Herpes Simplex Virus thymidine kinase TK promoter polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pCEP4 CAT Map of The figure below summarizes the features of the pCEP4 CAT vector The pCEP4 CAT nucleotide sequence for pCEP4 CAT is available for downloading from our website www
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