he DNA fragments eluted from the agarose gel were cloned into pGEM-TEasy vector which allows efficient sequencing using the sequencing primers for T7 and SP6 promoters. 3 )lL of eluted DNA (1 )lg/)lL) was ligated with 1 )lL ofpGEM-TEasy vector in the presence of 1 )lL of T4 DNA ligase in a 10 )lL reaction volume. The reaction was allowed to proceed at 4 oc for 16 h following which 8 )lL of the ligation mix was used to transform DH5-a strain of E.coli following standard protocols (9). The transformation mix was spread onto LB-agar plates containing appropriate ampicillin (100 )lg/mL) and the blue-white selection reagent (40 )lLiplate) (Sigma chemical company). The plate was incubated at 37°C for 12 h following which the white colonies were picked up for screening for presence of the gene of interest.

Sub-cloning ofPCR products into pGEM-TEasy vector