Xba I subcloning problem - (May/26/2008 )
Hi everyone!
I`ve tried to cloning a PCR product into a pBI121 vector, into XbaI-XhoI sites, and I`ve found positive clones by colony PCR. I`ve done minipreps and checked this clones whit diferents RE digestions, whit internals and externals restrictions sites, and in the all cases I`ve had beautiful bands with the correct size...excepting when I tried to digest whit XbaI again!!! 
I was tried everything! I did ligation and transformation again, whit a new PCR product digested whit a new XbaI...I used TOPO, pBluescript, pUC...always I have the same result: colony PCR positives, miniprepr RE digestions positives... excepting whit Xba. the site is gone! I don`t know what can I do! I`m going to sequence this clones, but at the moment, I can`t find a logical explanation!
Can anybody help me??? I`ll be very greatful!
what bacterial strain are you using? In some settings, the XbaI site will be methylated (if it is followed by TC), so this would explain why you cannot cut your plasmid with XbaI even if you could cut your (unmethylated) PCR product easily
GATC sequences in dam+ E. coli strains are methylated on the adenine. This methylation will inhibit cutting by XbaI when it overlaps the site. So, sites with sequence ....gaTCTAGA... or sequence ...TCTAGAtc... will fail to cut.
Hallo, this problen can by unique feature of sequence, it can tend to introduce mist mach by Taq polymerase. You can try hight fidelity polymerase like Pfu. I suggest you have designet primers with XbaI site on the 5 overhang. YOu cane use another primers with longer specer6bp behind restriction site
Good luck
Hi everyo
Hallo I have another explanation, Girl next lab has similar problem, she cloned pcr product sequenced it and found out, primers were mistmached, there was mistake in the primers. The company delivered bad primers
please replay to me results I am interested in
please replay to me results I am interested in
Baxa, I think my problem it`ll be resolve whit a strain dam-, but we`ve had problems whit bad primers too...in those cases, the company did it again, for free. If I can help you in anything, just tell me. And thanks for your answers too!
THANKS A LOT!!!! You have resolved my problem! I was using a primer whit the sequence TCTAGAtc...in a DH5-alfa E. coli strain!
THANKS!!!!