pcDNA3.1质粒图谱

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1、0 Invitrogenlife technologieswww.i nvitroge tech_service in vitroge n. compcDNA3.1(+)pcDNA3.1(-)Catalog nos. V790-20 and V795-20, respectivelyVersio n I 081401 28-0104iiTable of ContentsTable of Contents iiiImportant Information vPurchaser Notification viMethods 1Overview1Cloning into pcDNA3.12Trans

2、fection6Creation of Stable Cell Lines7Appendix 10pcDNA3.1 Vectors10pcDNA3.1/CAT12Technical Service13References15iiiivContentsShipp in g/StorageProductQualificati onImportant InformationpcDNA3.1 is supplied as follows:Catalog no.Con te ntsV790-2020 i g pcDNA3.1(+), lyophilized in TE, pH 8.020 i g pcD

3、NA3.1/CAT, lyophilized in TE, pH 8.0V795-2020 i g pcDNA3.1(-), lyophilized in TE, pH 8.020 i g pcDNA3.1/CAT, lyophilized in TE, pH 8.0Lyophilized plasmids are shipped at room temperature and should be stored at -20Each of the pcDNA3.1 vectors is qualified by restriction enzyme digestion with specifi

4、c restrictio n en zymes as listed below. Restricti on digests must dem on strate the correct banding pattern when electrophoresed on an agarose gel. The table below lists the restrictio n en zymes and the expected fragme nts.VectorRestriction EnzymeExpected Fragme nts (bp)pcDNA3.1(+)Nhe I5428Pst I13

5、56,4072Sac I109, 5319pcDNA3.1(-)Nhe I5427Pst I1363, 4064Sac I169, 5258pcDNA3.1/CATNhe I6217Pst I2145, 4072Sac I109, 6008Purchaser NotificationIn troduct ionUse of pcDNA3.1 is covered un der a n umber of differe nt lice nses as described below.CMV PromoterUse of the CMV promoter is covered un der U.S

6、. Pate nt Nos. 5,168,062 and 5,385,839 owned and lice nsed by the Uni versity of Iowa Research Fou ndati on and may be used for research purposes only . Commercial users must obtain a license to these patents directly from the Un iversity of Iowa Research Fou ndati on .In quiries for commercial use

7、should be directed to:Bre nda Ak insUn iversity of Iowa Research Fou ndati on (UIRF)214 Tech no logy Inno vati on Cen terIowa City, IA 52242Pho ne: 319-335-4549BGHPolyade ny lati onSig nalThe bov ine growth horm one (BGH) polyade ny lati on seque nee is lice nsed un der U.S.Pate nt No. 5,122,458 for

8、 research purposes only.“ Research purposes ” means usesdirected to the ide ntificati on of useful recomb inant prote ins and the in vestigati on of the recomb inant expressi on of prote ins, which uses shall in no eve nt in clude any of thefollowi ng:a. any use in humans of a CLAIMED DNA or CLAIMED

9、 CELL;b. any use in huma n of prote in or other substa nee expressed or made at any stage of itsproduction with the use of a CLAIMED DNA or a CLAIMED CELL;c. any use in which a CLAIMED DNA or CLAIMED CELL would be sold ortransferred to another party other than Invitrogen, its AFFILIATE, or its SUBLI

10、CENSEE;d. any use in conn ecti on with the expressi on or producti on of a product inten ded for sale or commercial use; ore. any use for drug scree ning or drug developme nt.In quiries for commercial use should be directed to:Bennett Cohen, Ph.D.Research Corporation Tech nologies101 North Wilmot Ro

11、ad, Suite 600Tucso n, AZ 85711-3335Tel: 1-520-748-4400Fax: 1-520-748-0025#MethodsOverview1#In troduct ionpcDNA3.1(+) and pcDNA3.1(-) are 5.4 kb vectors derived from pcDNA3 and designed for high-level stable and tran sie nt expressi on in mammalia n hosts. High-level stable and non-replicative transi

12、ent expression can be carried out in most mammalian cells. The vectors contain the following elements:?Huma n cytomegalovirus immediate-early (CMV) promoter for high-level expressi onin a wide range of mammalia n cells? Multiple cloning sites in the forward (+) and reverse (-) orientations to facili

13、tate clo ning?Neomyc in resista nee gene for selecti on of stable cell li nes? Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T an tigen (e.g. COS-1, COS-7)The control plasmid, pcDNA3.1/CAT, is included for use as a positive control for tran s

14、fecti on and expressi on in the cell li ne of choice.Experime ntalOutli neUse the following outline to clone and express your gene of interest in pcDNA3.1.1. Consult the multiple cloning sites described on pages 3-4 to design a strategy to clone your gene into pcDNA3.1.2. Ligate your insert into the

15、 appropriate vector and transform into E. coli. Selecttransformants on LB plates containing 50 to 100pg/ml ampicillin.3. An alyze your tra nsforma nts for the prese nee of in sert by restricti on digesti on.4. Select a tran sforma nt with the correct restricti on patter n and use seque ncing to con

16、firm that your gene is cloned in the proper orie ntati on.5. Tran sfect your con struct into the mammalia n cell line of in terest using your own method of choice. Gen erate a stable cell li ne, if desired.Test for expressi on of your recomb inant gene by wester n blot an alysis or fun cti onal assa

17、y.#Cloning into pcDNA3.1In troduct ionDiagrams are provided on pages 3-4 to help you design a cloning strategy for ligating your gene of interest into pcDNA3.1. General considerations for cloning and transformation are listed below.Gen eral MolecularBiologyTech niq uesFor help with DNA ligations,E.

18、coli transformations, restriction enzyme analysis,purificati on of sin gle-stra nded DNA, DNA seque ncing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual(Sambrook et al., 1989) or CurrentProtocols in Molecular Biology (Ausubel et al., 1994).E. coli StrainMany E. coli st

19、rains are suitable for the propagation of this vector including TOP10F?RDH5 a -T1 , and TOP10. We recommend that you propagate vectors containing inserts inE. coli stra ins that are recomb in ati on deficie nt ( recA) and endon uclease A-deficie nt (endA).For your convenien ce, TOP10F is available a

20、s chemically compete nt or electrocompete ntcells from In vitroge n.ItemQua ntityCatalog no.?One Shot TOP10F (chemically compete nt cells)21 x 50卩1C3030-03?Electrocomp ? TOP10F 5 x 80卩1C665-55? ,Ultracomp TOP10F(chemically compete nt cells)5 x 300卩1C665-03Tran sformatio nMethodYou may use any method

21、 of your choice for tran sformati on. Chemical tra nsformati on is the most convenient for most researchers. Electroporation is the most efficient and the method of choice for large plasmids.Maintenance of pcDNA3.1To propagate and maintain pcDNA3.1, we recommend resuspending the vector in 20 sterile

22、 water to make a 1卩 g/ 卩 l stock solution. Store the stock solution at -20Use this stock solution to tran sform a recA, en dA E. coli strain like TOP10F”DH5RT1 , TOP10, or equivale nt. Select tra nsforma nts on LB plates containing 50 to100 卩 g/ml ampicillin. Be sure to prepare a glycerol stock of y

23、our plasmid-containing E. coli strain for long-term storage (see page 5).11Clo ningCon siderati onspcDNA3.1(+) and pcDNA3.1(-) are non fusi on vectors. Your in sert must contain a Kozak tran slati on in itiati on seque nee and an ATG start cod on for proper in itiati on of tran slati on (Kozak, 1987

24、; Kozak, 1991; Kozak, 1990). An example of a Kozak consen sus seque nce is provided below. Please note that other sequences are possible (see references above), but the G or A at position -3 and the G at position +4 are the most critical for function (show n in bold). The ATG in itiati on cod on is

25、show n un derl in ed.(G/A) NNATG GYour in sert must also contain a stop codo n for proper term in ati on of your gene. Please note that the Xba I site contains an internal stop codo n (TCTAGA).contin ued on n ext page3Cloning into pcDNA3.1, continuedMultiple Clo ningSite of pcDNA3.1(+)Below is the m

26、ultiple cloning site for pcDNA3.1(+). Restriction sites are labeled to in dicate the cleavage site. The Xba I site contains an in ternal stop cod on (TCTAGA). The multiple cloning site has bee n con firmed by seque ncing and fun cti onal test ing.Thecomplete sequence of pcDNA3.1(+) is available for

27、downloading from our web site ( ) or from Technical Service (see page 13).For a map and adescription of the features of pcDNA3.1(+), please refer to the Appendix , pages 10-11.enhancer region (3end)689 CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCGCAATTATAIIII749 TAACAACTCC GCCCCA

28、TTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT3 end of hCMVputative tran scripti onal start809 AAGCAGAGCT CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATACT7 promoter/primer binding siteNhe IPme I Afl II Hind III Asp718 I Kpn I一iIIIIII869 GACTCACTAT AGGGAGACCC AAGCTGGCTA GCGTTTAAAC TTAAGCTTGG

29、 TACCGAGCTCBamH IBstX I* EcoR IEcoR VBstX I* Not IXho I929GGATCCACTA GTCCAGTGTG GTGGAATTCT GCAGATATCC AGCACAGTGG CGGCCGCTCGpcDNA3.1/BGH reverse priming site989Xba IApa I Pme IAGTCTAGAGG GCCCGTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC1049 CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT GGAAGGTGCC

30、ACTCCCACTGBGH poly (A) siteIi1109TCCTTTCCTA ATAAAATGAG GAAATTGCATPlease note that there are two BstX I sites in the polylinker.contin ued on n ext page#Cloning into pcDNA3.1, continuedMultiple Clo ning Site of pcDNA3.1(-)Below is the multiple cloning site for pcDNA3.1(-). Restriction sites are label

31、ed to in dicate the cleavage site. The Xba I site contains an in ternal stop cod on (TCTAGA). The multiple cloning site has bee n con firmed by seque ncing and fun cti onal test ing.Thecomplete sequence of pcDNA3.1(-) is available for downloading from our web site ( ) or from Technical Service (see

32、page 13). For a map and a description of the features of pcDNA3.1(-), please see the Appendix , pages 10-11.enhancer region (3end)689 CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG.CAAT. TATAIIi i749 TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT3 end of hCMVput

33、ative transcriptional start809 AAGCAGAGCT CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATACT7 promoter/primer binding siteNhe IPme IApa I Xba I Xho I Not I 1|Tii|869 GACTCACTAT AGGGAGACCC AAGCTGGCTA GCGTTTAAAC GGGCCCTCTA GACTCGAGCGBstX I* EcoR VEcoR IBstX I*BamH I929 GCCGCCACTG TGCTGGATAT CTGCA

34、GAATT CCACCACACT GGACTAGTGG AtCCGAGCTCAsp718 I Kpn I Hin d III Afl II Pme IrcDNAB.WBGH reverse priming site989 GGTACCAAGC TTAAGTTTAA ACCGCTGATC AGCCTCGACT GTGCCTTCTA GTTGCCAGCC1049 ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC CTTGACCCTG GAAGGTGCCA CTCCCACTGTBGH poly (A) siteII1109 CCTTTCCTAA TAAAATGAGG AAATTGCA

35、TC*Please note that there are two BstX I sites in the polylinker.contin ued on n ext pageCloning into pcDNA3.1, continuedE. coli Tran sformatio nTran sform your ligati on mixtures into a compete ntrec A, endA E. coli strain (e.g. TOP10F?RDH5 a -T1 , TOP10) and select tran sforma nts on LB plates con

36、 tai ning 50 to 100ampicillin. Select 10-20 clones and analyze for the presence and orientation of your insert.V-We recomme nd that you seque nce your con struct with the T7 Promoter and BGH Reverse primers (Catalog nos. N560-02 and N575-02, respectively) to con firm that your gene is in the correct

37、 orie ntati on for expressi on and contains an ATG and a stop codo n. Please refer to the diagrams on pages 3-4 for the sequences and location of the priming sites. The primers are available separately from In vitroge n in 2pg aliquots.Prepari ng a Glycerol StockOnce you have identified the correct

38、clone, purify the colony and make a glycerol stock for long-term storage. You should keep a DNA stock of your plasmid at -20 C.? Streak the original colony out on an LB plate containing 50pg/ml ampicillin. Incubatethe plate at 37 C overnight.? Isolate a single colony and inoculate into 1-2 ml of LB

39、containing 50pg/ml ampicillin.?Grow the culture to mid-log phase (OD 600 = 0.5-0.7).?Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and tra nsfer to a cryovial.? Store at -80 C.TransfectionIn troduct ionOnce you have verified that your gene is cloned in the correct orientation and contains

40、an in itiati on ATG and a stop cod on, you are ready to tra nsfect your cell line of choice. We recomme nd that you in clude the positive con trol vector and a mock tran sfecti on (n egative con trol) to evaluate your results.Plasmid Preparati onPlasmid DNA for tran sfectio n into eukaryotic cells m

41、ust be clea n and free from phe nol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids decreas ing tran sfectio n efficie ncy. We recomme nd isolati ng plasmid DNA using the? ?S.N.A.P.MiniPrep Kit (10-15pg DNA, Catalog no. K1900-01), the S.N.A.P.MidiPrep Kit (

42、10-200 旳 DNA, Catalog no. K1910-01), or CsCl gradient centrifugation.Methods ofTran sfectio nFor established cell li nes (e.g. HeLa), please con sult origi nal referen ces or the supplier of your cell line for the optimal method of transfection. We recommend that you follow exactly the protocol for

43、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 tra nsfecti on in clude calcium phosphate (Che n and Okayama,

44、 1987; Wigleretal., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989) and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). Invitrogen offers the Calcium Phosphate Transfection Kit (Catalog no. K2780-01) and a large selection of reage nts for tran sfecti on. For mo

45、re in formati on, please refer to our World Wide Web site ( ) or call Technical Service (see page 13).Positive Con trolpcDNA3.1/CAT is provided as a positive con trol vector for mammalia n tra nsfecti on and expression (see page 12) and may be used to optimize transfection conditions for your cell l

46、ine. The gene encoding chloramphenicol acetyl transferase (CAT) is expressed in mammalia n cells un der the con trol of the CMV promoter. A successful tra nsfecti on will result in CAT expressi on that can be easily assayed (see below).Assay for CAT ProteinYou may assay for CAT expressi on by ELISA

47、assay, western blot an alysis, fluorometric assay, or radioactive assay (Ausubel et al., 1994; Neumann et al., 1987). If you wish to detect CAT protein using western blot an alysis, you may use the An ti-CAT An tiserum (Catalog no. R902-25) available from Invitrogen. Other kits to assay for CAT prot

48、ein using ELISA assay are available from Roche Molecular Biochemicals (Catalog no. 1 363 727) and Molecular Probes (Catalog no. F-2900).7Creation of Stable Cell LinesIn troduct ionThe pcDNA3.1(+) and pcDNA3.1(-) vectors contain the n eomyc in resista nee gene for selection of stable cell lines using

49、 neomycin (Geneticin ? ). We recommend that you test the sen sitivity of your mammalia n host cell to Gen etic in ? as n atural resista nee varies among cell li nes. Gen eral in formati on and guideli nes are provided in this secti on for your convenien ce.?Gen etici nSelectiveAn tibiotic?Gen etic i

50、n Selective An tibiotic blocks protein syn thesis in mammalia n cells by in terfer ing with ribosomal fun cti on. It is an amino glycoside, similar in structure to n eomyc in, gen tamyc in, and kan amyc in. Expressi on of the bacterial amino glycoside phosphotra nsferase gene (APH), derived from Tn

51、5, in mammalian cells results in detoxification of Geneticin (Southern and Berg, 1982).Geneticin ? Selectio n Guideli nes?Ge neticin Selective An tibiotic is available from In vitroge n (Catalog no. 10486-025).Use as follows:? Prepare Gen eticin in a buffered solution (e.g. 100 mM HEPES, pH 7.3).? U

52、se 100 to 800 pg/ml of Geneticinin complete medium.? Calculate concen trati on based on the amou nt of active drug (check the lot label).? Test varyi ng con ce ntratio ns of Ge neticin on your cell line to determi ne the concen trati on that kills your cells (see below). Cells differ in their suscep

53、tibility to Geneticin ?.?Cells will divide once or twice in the presence of lethal doses of Geneticin, so the effectsof the drug take several days to become apparent. Complete selection can take up to 3 weeks of growth in selective media.Determ in ation ofAn tibioticSen sitivityTo successfully gen e

54、rate a stable cell li ne express ing your gene of i nterest from pcDNA3.1, you need to determine the minimum concentration of Geneticin? required tokill your un tra nsfected host cell li ne. We recomme nd that you test a range of concen trati ons to en sure that you determ ine the mini mum concen tr

55、ati on n ecessary for your host cell li ne.1. Plate or split a con flue nt plate so the cells will be approximately 25% con flue nt. Prepare a set of 7 plates. Allow cells to adhere over ni ght.2. The n ext day, substitute culture medium with medium containing vary ing? ? concentrations of Geneticin

56、(0, 50, 100, 200, 400, 600, 800 g/ml Geneticin ).3. Reple nish the selective media every 3-4 days, and observe the perce ntage of surviv ing cells.4. Count the number of viable cells at regular intervals to determine the appropriate?concen trati on of Gen etic inthat preve nts growth with in 2-3 wee

57、ks after additi on ofGeneticin ?.Creation of Stable Cell Lines, continuedPossible Sites for Lin earizati on of pcDNA3.1(+)Prior to tran sfecti on, we recomme nd that you lin earize the pcDNA3.1(+) vector.Linearizing pcDNA3.1(+) will decrease the likelihood of the vector integrating into the genome i

58、n a way that disrupts the gene of in terest or other eleme nts required for expressi on in mammalian cells. The table below lists unique restriction sites that may be used to lin earize your con struct prior to tran sfecti on. Other uni que restricti on sites are possible. Be sure that your insert d

59、oes not contain the restriction enzyme site you wish to use to lin earize your vector.En zymeRestriction Site (bp)Locati onSupplierBgl II12Upstream of CMV promoterInvitrogen, Catalog no. 15213-028Mfe I161Upstream of CMV promoterNew En gla nd BiolabsBst1107 I3236End of SV40 polyA*AGS , Fermentas, Tak

60、ara, Roche Mol. BiochemicalsEam1105 I4505Ampicilli n gene*AGS , Fermentas, TakaraPvu I4875Ampicilli n geneInvitrogen, Catalog no. 25420-019Sca I4985Ampicilli n geneInvitrogen, Catalog no. 15436-017Ssp I5309bla promoterInvitrogen, Catalog no. 15458-011*Angewandte Gentechnologie SystemePossible Sites

61、for Lin earizati on of pcDNA3.1(-)The table below lists unique restriction sites that may be used to linearize your pcDNA3.1(-) con struct prior to tra nsfecti on.Other uni que restricti on sites are possible.Be sure that your insert does not contain the restriction enzyme site you wish to use to li

62、n earize your vector.En zymeRestriction Site (bp)Locati onSupplierBgl II12Upstream of CMV promoterInvitrogen, Catalog no. 15213-028Mfe I161Upstream of CMV promoterNew En gla nd BiolabsBst1107 I3235End of SV40 polyA*AGS , Fermentas, Takara, Roche Mol. BiochemicalsEam1105 I4504Ampicilli n gene*AGS , Fermentas, TakaraPvu I4874Ampicilli n geneInvitrogen, Catalog no. 25420-019Sca I4984Ampicilli n geneInvitrogen, Catalog no. 15436-017Ssp I5308bla promoterInvitrogen, Catalog no. 15458-011*Angewandte Gentechnologie Systemecontin ued on n ext page9Creation of Stable Cell