Double digestion and ligation problem - (May/31/2010 )
I designed primers with BahH1, XhoI site, double digested vector with same enzymes with common Buffer (Roche buffer B, enzymes are from the same company), Digested PCR amplified fragment with same enzymes, Gel purified (checked vector and insert with control to ensure proper digestion), Ligated 1:3 vector insert ratio, transformed, colonies were appeared in selection media, did colony PCR, amplified and got right size, purified plasmid, restriction enzymes did not cut out, did again PCR using plasmid as template amplified well,..Empty vector also amplified with gene specific primers (Two genes…corresponding size), got wrong sequence in sequencing result, I appreciate your suggestions..
i've found that positive colony pcr clones are not always correct so i usually just test digests in the first place to identify positives. sometimes i have to do it many many many times to find a positive clone. just keep testing colonies.