I have a problem with cloning pHannibal cassette in pART27vector....I cut phannibal cassette and open pART27 with NotI and seems to be OK....after i treat the pART27 with CIAP, extract all the two bands from agarose gel...ligate and cloning with eclectroporation, the ligation in E.coli DH5alfa...after i select on antibiotic Kanamicin 50ug/ml...but not works....There's anyone that can help me????

Vinny:

See my reply to your similar post in the Plasmid/Cosmid/BAC/PAC topic of the molecular biology forum:

http://www.scientistsolutions.com/t3569-Problem+with+pART27+vector.html

Have you tried lower concentrations of antibiotics during your selection step. Also, have you confirmed any surviving culture after your electroporation step. It may be possible that your culture is no longer viable. In such case, you may need to repeat this cloning step.

Tony Rook

Thank you very much for the reply.....
I try to apply your suggestion.... let we see...

I try with go down Kanamicyn antibiotic concentration 25ug/ml but nothing....No colony....I have also a negative control with ciapped vector in ligation without fragment and i not have colonies.....the same bacterial concentration on LB without antibiotic show a good growth and the trasformation with the uncutted vector work.....
It's very strange....my new idea is to not purify the vector on agarose gel after NOT I and Ciap treatment but purify or with Kit for purification of PCR products or with phenol-chloroform because probably there are some problems with residual presence of agarose or ethidium bromide ....i don't know