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)
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AD2: 5' NGT CGA (G/C)(A/T)G ANA (A/T)GA A 3' 、
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OPA-2: 5' TGC CGA GCTG 3'
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T-DNA left border primer sequences (T-DNA left border sequences (working dilution: 50uM)
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TL-1: 5' CAG CCA ATT TTA GAC AAG TAT CA 3'
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TL-2: 5' AAC TGT AAT GAC TCC GCG CAA TA 3'
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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.
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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
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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: to, and then
Cycling 92oC; 5 min;[Add 0.2 ul Taq polymerase (Perkins-Elmer's or Stratagene's) after 4 min denaturation, link to clcling
Cycling (5 cycles of the following):
94oC, 1'
62oC, 1'
72oC2.5'
Cycling (1 cycle of the following):
94oC, 1'
25oC, 3'
Ramp to 72oC in 3'
72oC2.5'
Cycling (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
72oC, 5'
Cycling
4oC. soak
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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
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Thermo-cyclings (0.2u. Taq polymerase was added after 4 min denaturation) to, and then.
Cycling for 12 cycles:
94oC, 30''
64oC, 1'
72oC, 2.5'
94oC,30''
64oC, 1'
72oC, 2.5'
94oC, 30''
44oC, 1'
72oC, 2.5'
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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
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Thermo-cycling: (0.2ul Taq polymerase was added after 4' denaturation in #10), and then .
Thermo-cycling for 25 cycles:
94oC,1'
37oC, 1
72oC, 1'
1.5% agarose electrophoresis of all three PCR reaction products.
Cloning PCR product by using Stratagene's PCR polishing kit and pCRScript Amp (SK+) cloning kit or my favorite one-tube cloning method .
Purify the PCR products using Pharmacia's Spin-Column S-300.