puc19 vector - (Dec/14/2007 )
I am currently having a big problem with my puc19 vector which i need to do a double digestion for my ligation.
I tried restricting with SacI and XbaI enzymes (both single and double digestions), and i found that both sites were not working at all (i.e. i can't manage to cut the vector at all). I have tried restricting another plasmid (pBI121) with the same set of enzymes and buffer at the same temperature ans time, and it works great. Also, i have tried digesting the same puc19 with SacI and XbaI for several times, but it is still a failure.
However, all the puc19 transformed colonies which i inoculated were blue in colour on the AXI plate. Is there a possibility for the puc19 vector to loose both the SacI and XbaI restricted sites on itself?
Need desperate help!!
Where did you get the vector from? Is it new? If it isn't maybe it's been already used by someone who somehow deleted that area while cloning.
Try to sequence that area to check if the restriction sites are still there.