Ok, I am running out of ideas. I am attempting to clone a fragment of a gene from potato and sequence it in attempts to design better primers to amplify the whole gene (promoter included). I can amplify the fragment pretty regularly using a platinum supermix PCR solution. I then isolate the fragment from a gel using a mo-bio kit. Then I A-tail the fragment, ligate into Pgem T-easy vector, transform via heat-shock, and plate on IPTG x-gal blue selective media. I got 5 white colonies (out of 100+ blue colonies). I performed PCR on the white colonies using T7 and SP6 primers (located on T-easy) This showed an insertion of appropriate length (3.5Kb) So then I grew the ecoli out, and isolated the plasmid, and sent it in to be sequenced using the T7 and SP6 primers. The sequencing returned back only vector sequence... And so to double check that the insert was truly in the plasmid, I ran a digestion using EcoRI. ran the results out on a gel, and observed only one band at 2.5Kb (supercoiled vector?) So, everything points to my insert being present, until I sequence or digest.
Is it possible that the colony PCR is amplifying the vector, and giving me a false positive? (if so, why is the colony still white when grown on IPTG x-gal plates?) Can someone offer any type of conclusion here? I am really stumped. Thanks
p.s. what a great idea for a forum.
Edited by nimrod337, 03 August 2010 - 12:41 PM.