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