Hi, I want to know, how can we know that our vector is self ligated? I did single restriction digestion of both vector and insert with Hind III, after the transformation, we obtained only 4 colonies and all colonies were negative in the colony PCR. All 4 colonies showed plasmid self-ligation. Could someone help us?
Dear Nulu
did you performed phosforilation with CIP and gel extraction of the digested plasmid after digestion?
Gel extraction is important to avoid the insert re-ligation and remove the not digested plasmid that sometimes also if not visible on gel may give colonies expecially if you working with cells with high competency
Treatment with alkaline phospatase ( https://international.neb.com/products/m0290-alkaline-phosphatase-calf-intestinal-cip#Product%20Information ) prevent the religation of the linearized vector.
ciao
Manuele
You should load your digests on an agaorse gel, cut out the correct bands and extract the DNA from there. This way you get rid of undigested plasmid.
Additionaly SAP/CIP phospotylation will prevent self ligation.
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