TAIL-PCR to clone flanking sequence from Feldmann's lines

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 to #18, and then #19, #20, #21, #53

Cycling #10: 92^oC; 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):

94^oC, 1'
62^oC, 1'
72^oC2.5'

Cycling #19 (1 cycle of the following):

94^oC, 1'
25^oC, 3'
Ramp to 72^oC in 3'
72^oC2.5'

Cycling #20 (15 cycles of the following):

94^oC, 30''
68^oC, 1'
72^oC, 2.5'

94^oC, 30''
68^oC, 1'
72^oC; 2.5'
94^oC, 30''
44^oC; 1'
72^oC; 2.5'

cycling #21:

72^oC, 5'

Cycling #53

4^oC. soak

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) to #22, and then #21, #53. Cycling #22 for 12 cycles:

94^oC, 30''
64^oC, 1'
72^oC, 2.5'

94^oC,30''
64^oC, 1'
72^oC, 2.5'

94^oC, 30''
44^oC, 1'
72^oC, 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), and then #12, #52 . Thermo-cycling #12 for 25 cycles:

	94^oC,1'
	37^oC, 1
	72^oC, 1'

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 .

Reference:

	Liu YG, Mitsukawa N, Oosumi T, Whittier RF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 1995 Sep;8(3):457-463.
	Chun-Ming Liu, John McElver, Irris Tzafrir, Ronny Joosen, Peter Wittich, David Patton, Andre A.M. van Lammeren and David Meinke (2002) Condensin and cohesin knockouts in Arabidopsis exhibit a titan seed phenotype . Plant Journal, 29: 405-415. [PDF file]

	AD2: 5' NGT CGA (G/C)(A/T)G ANA (A/T)GA A 3'
	OPA-2: 5' TGC CGA GCTG 3'