Dear All,
I have digested my vector with ApaI and my PCR product is flanked by ApaI site, which I use to cut the insert from pGEM T vector after subcloning. We use CIP to prevent self ligation of Vector. Can my insert be self ligated ? I have three inserts. 900 bp, 2200 bp and 1700 bp. Theoretically, these inserts which are flanked by ApaI site can be self ligated, giving rise to longer fragment before getting ligated into destination vector. Is it possible? or, I am just over thinking? How to deal with this issue? I assume I do not need to add CIP in the restriction digestion mixture of pGEMT-my gene.
Göran Hjälm
Thank you very much for your valuable input.It helps a lot
Hi, I think self ligation of your fragment is possible because it has only one G overhang. You can work without CIP and after ligation and transformation check your fragment size by PCR and Digestion. Between your colonies there will be appropriate colony. Before the ligation you should inactive the restriction enzyme by heating according manufacturer protocol. I did not use CIP and the results of the ligation was good.
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