TAIL-PCR to clone flanking sequence from Feldmann's lines
Based on Liu et al, Plant J. 8:457 with modifications
Comments or Questions? Send to me at:chun-mingliu@plant.wag-ur.nl
The first thing you need to do before starting your TAIL-PCR is to figure out
which border of the T-DNA is flanking the plant genome, especially with Ken Feldmann's
lines since the concatameric nature of the T-DNA inserts. The best way to do that is to
follow the procedure designed by Ponce et al, which was published in the Plant Journal
[Ponce et al,(1998): Rapid discrimination of sequences flanking and within T-DNA insertions in the Arabidopsis
genome. The Plant Journal, 14:497-501.
Primer sequences (working dilution: 50uM)
- AD2: 5' NGT CGA (G/C)(A/T)G ANA (A/T)GA A 3'
- OPA-2: 5' TGC CGA GCTG 3'
T-DNA left border primer sequences (T-DNA left border sequences
(working dilution: 50uM)
- TL-1: 5' CAG CCA ATT TTA GAC AAG TAT CA 3'
- TL-2: 5' AAC TGT AAT GAC TCC GCG CAA TA 3'
- TL-3: 5' TCT GGG AAT GGC GTA ACA AAG GC 3'
- Genomic DNA extraction (CTAB method was used in my case, but it might be not essential).
- Dilute the genomic DNA to 20ng/ul with water.
- To ease the pipetting procedure, a PCR stock solution was used (I found both the
Perkin-Elmer's and Stratagene's Taq polymerases work very well, in condition of using their own
buffers):
- 705ul water
- 100ul 10X Taq polymerase buffer (Perkin's or Stratagene's)
- 20ul 10mM dATP
- 20ul 10mM dCTP
- 20ul 10mM dGTP
- 20ul 10mM dTTP
- Primary reaction mixture for the TAIL-PCR
- 44ul PCR stock
- 2.5ul TL-3
- 2.5ul AD-2 or OPA-2
- 1ul Genomic DNA (20ng/ul)
- Thermo-cycling: #10 L #18 L #19 L #20 L#21 L #53
- Cycling #10: 92oC; 5 min;[Add 0.2 ul Taq polymerase (Perkins-Elmer's or Stratagene's)
after 4 min denaturation, link to clcling #18
- Cycling #18 (5 cycles of the following):
- 94oC, 1'
- 62oC, 1'
- 72oC2.5'
- Cycling #19 (1 cycle of the following):
- 94oC, 1'
- 25oC, 3'
- Ramp to 72oC in 3'
- 72oC2.5'
- Cycling #20 (15 cycles of the following):
- 94oC, 30''
- 68oC, 1'
- 72oC, 2.5'
- 94oC, 30''
- 68oC, 1'
- 72oC; 2.5'
- 94oC, 30''
- 44oC; 1'
- 72oC; 2.5'
- cycling #21:
- Cycling #53
- Dilute the primary reaction product with water to 1/50, then set up the 2nd reaction
- 44ul PCR stock
- 2.5ul TL-2 (50uM)
- 2.5ul AD-2 or OPA-2 (50uM)
- 1ul 1/50 diluted 1st reaction
- Thermo-cyclings: #10 (0.2u. Taq polymerase was added after 4 min denaturation)
L #22 L #21 L #53.
Cycling #22 for 12 cycles:
- 94oC, 30''
- 64oC, 1'
- 72oC, 2.5'
- 94oC,30''
- 64oC, 1'
- 72oC, 2.5'
- 94oC, 30''
- 44oC, 1'
- 72oC, 2.5'
- Dilute the 2nd PCR product to 1/50 with water, and carry out the tertiary reaction
- 44ul PCR stock
- 2.5ul TL-1
- 2.5ul AD-2 or OPA-2 (50uM)
- 1ul 1/50 diluted 2nd reaction product
- Thermo-cycling: #10 (0.2ul Taq polymerase was added after 4' denaturation in #10) L #12 L #52 .
Thermo-cycling #12 for 25 cycles:
- 1.5% agarose electrophoresis of all three PCR reaction products.
- Purify the PCR products using Pharmacia's Spin-Column S-300.
- Cloning PCR product by using Stratagene's
PCR polishing kit and pCRScript Amp (SK+) cloning kit or my favorite one-tube cloning method .
