Target Discovery and Validation Reviews and Protocols

72 Beaty et al.
4. Phenol:cholorform extract with 240 µL of PC8 (480 µL total volume).
5. Vortex, centrifuge for 5 min at 21,000g at 4°C.
6. Remove 200 µL of the upper aqueous phase containing the nucleic acids and
aliquot into the “ligase-plus” tube; aliquot the rest of the aqueous upper phase into
“ligase-minus” tube.
7. Equalize the volume of the plus and minus ligase samples by adding 160 µL of
LoTE to the ligase-minus sample. EtOH precipitate both the plus and minus tubes
with 7.5 M ammonium acetate.
8. Centrifuge for 30 min at 11,000g at 4°C.
9. Remove the supernatant and wash twice with 200 µL of 75% EtOH.
10. Resuspend the ligase-plus reaction in 5 µL of LoTE.
11. Resuspend the ligase minus reaction in 3 µL of LoTE.
3.2.7. Ligate to Form Ditags
Using high concentration T4 ligase (5 U/µL), set up a ligation to form ditags.
1. Add 5 µL of 2X ligase-plus mix to the ligase-plus tube.
2. Add 3 µL of 2X ligase-minus mix to the ligase-minus tube. Therefore, the total
volume of ligase-plus is 10 µL and of ligase-minus is 6 µL.
3. Incubate overnight at 16°C.
4. Add 10 µL of LoTE to the ligase-plus tube (20 µL total volume).
5. Add 14 µL of LoTE to the ligase-minus tube (20 µL total volume).
3.2.8. PCR Amplification of Ditags
Use the P1 and P2 primers (350 ng/µL each) to amplify ditags for a test PCR
to determine the optimal conditions for the large-scale PCR that follows.
1. Optimize amplification by using different dilutions of the ditag template (see Note
18). Cycle conditions are as follows: (A) 1 cycle: 94°C for 1 min; (B) 27 cycles
(ligase-plus) or 35 cycles (ligase-minus): 94°C for 30 s; 55°C for 1 min; 70°C for
30 s; and (C) 1 cycle: 7°C for 5 min.
2. Use 27 cycles for the ligase-plus reactions, and 35 cycles for the ligase-minus
reactions (see Note 19).
3. Remove 10 µL from each reaction and mix with 1 µL of loading dye. Electrophorese
the samples on a 1-mm 12% polyacrylamide gel. Use 10- and 100-bp
ladder as a marker. Amplified ditags should be 102 bp. A background band of
equal or lower intensity occurs around 80 bp. All other background bands should
be of substantially lower intensity. The ligase-minus samples should not contain
any amplified product of the size of the ditags even at 35 cycles.
After the PCR test to determine the appropriate dilution, perform a large-scale
PCR. About 300 reactions of 50 µL each of the optimal dilution is needed for
the pooled PCR products. Three 96-well plates with a 50-L reaction per well is
adequate. Use one less cycle for the large-scale than for the small-scale PCR.