Fig. 2.

PCR assays to verify the insert of each colony. The PCR amplified subtracted cNDA was ligated into the PCR 2·1 TA cloning vector, and transformed into DH5α competent cells. The transformed DH5α competent cells were grown overnight at 37 °C on LB agar plates containing ampicillin, X-gal, and isopropyl-β-D-thiogalactopyranoside for white/blue colony selection. White colonies were isolated and grown individually overnight at 37 °C in 200 µl of LB broth containing ampicillin. The insert of each colony was PCR amplified using a 1-µl aliquot of the bacteria growth. The size of inserts showing in this example varied from about 350 base pair (bp) to about 1100 bp. Lanes 7 and 10 showed that bacteria colonies had more than one plasmid. Lanes 2 and 14 showed no insert in these colonies. Lane 20 is 100 bp DNA markers.