| 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)
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