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Monday, November 10, 2003
Description Inverse PCR Protocol This method can be used directly for pBIN19 or pBI101 based T-DNA inserts Procedure Digestion reaction: 2.5g genomic DNA (CTAB) 1x 10x enzyme buffer 20-40U restriction enzyme xl ddH2O (as required to balance the reaction) Set up 1 reaction per enzyme Digest at 37C for 2 hours PCI CLEANUP Adjust reaction volume to 100l with ddH2O Add 100l PCI (1x sample volume; Phenol:Chloroform:Isoamyl alcohol, 25:24:1) Vortex for 3 min, centrifuge at 15,000 rpm for 5 min at 15C Transfer sup (95l) to new tube and add 95l PCI (1x sup volume) Vortex for 3 min, centrifuge at 15,000 rpm for 5 min at 15C Transfer sup (90l) to new tube and add 90l Chloroform (1x sup volume) Vortex for 3 min, centrifuge at 15,000 rpm for 5 min at 15C Transfer sup (80l) to new tube, add 8l 3M NaOAc and 200l 100% EtOH Place at -20C for 20 min then centrifuge at 15,000 rpm for 20 min at 4C Discard sup, wash pellet with 500l 70% EtOH Dry pellet and resuspend in 25l ddH2O Ligation Adjust digested sample to 40ng/l. If 2.5g was used for the digestion, you can assume the sample is this concentration. If a different initial amount was used you may want to quantify the sample Add 5l of the sample to 31l of ligation solution A (Takara, Kyoto, Japan). DNA concentration is 1-10ng/l) Add 4l of ligation solution B and incubate sample at 16C for at least 2 hrs (overnight is also okay) EtOH PRECIPITATION Add 4l 3M NaOAc and 100l 100% EtOH Place at -20C for 20 min then centrifuge at 15,000 rpm for 20 min at 4C Discard sup, wash pellet with 500l 70% EtOH (pellet is not visible) Dry pellet and resuspend in 25l ddH2O PCR Amplification 1x Reagent 2.0l 10x ExTAQ Buffer 2.0l 2.5mM dNTPs 0.8l 10M Binv1 primer (or Binv4) 0.8l 10M Binv2 primer (or Binv5) 0.2l ExTAQ 13.2l ddH2O 1.0l ligated sample PCR Cycle 1x 3 min 96C 25x 30 sec 96C, 30 sec 56C, 1 min 72C 1x 5 min 72C Prepare IPCR reactions with either the Binv1/Binv2 (right border) or Binv4/Binv5 (left border) primer set Run on 2% Seakem gel and extract putative IPCR bands Check / Sequence Quantify extracted bands. If unsure about quality or quantity of the IPCR bands, re-amplify by PCR Successful sequencing can be done with as little as 1.5 to 3.0ng/l. Typical reactions have 5l sample, 2l BigDye, 2l 1/2 BigDye and 1l 3.2M primer Use Binv2 or Binv4 as the sequencing primer, depending on which primer set was used for the PCR Recipes Supplies For IPCR from right border: Binv1 primer: amplifies into T-DNA (5'-ACAGAACCGCAACGTTGAA) Binv2 primer: amplifies out of T-DNA (5'-TCGTTTCCCGCCTTCAGTTTA) Restriction enzymes: AluI, HhaI, NheI, SspI, PstI, PvuII For IPCR from left border: Binv4 primer: amplifies out of T-DNA (5'-TAAAAACGTCCGCAATGTGT) Binv5 primer: amplifies into T-DNA (5'-AGTGAGACGGGCAACAGCT) Restriction enzymes: AluI, HhaI, HinfII, PvuII Tips (责任编辑：泉水) |
Inverse PCR Protocol
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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