Hi all,
I have been trying to do some cloning recently. When I am doing the ligation, I have 1 control: No insert control. In the no insert control, I did not put insert into the vector, which is also treated with Alkaline Phosphatase. Therefore, in this control, there should have no colonies formed. However, there are almost as much colonies formed as the true ligation, i.e. with both insert and vector.
Therefore, is there reason why and how can I avoid this problem?
Did you check that your vector was properly digested ? If there is still a significant fraction of the vector that is not digested at all, it will be insensitive to alkaline phosphatase and will transform very efficiently.
Pierre Béguin After I did the double digestion, I realised that most of the time, there are a lot of smear between the vector and the insert. I prepared 3mg of DNA and used Thermo Scientific fast digest to cut out NotI and BamHI. I put it in the incubator for 37 degrees for 15 mins. Why do I get is many smear bands and how do I remove it?
Thanks?
Mathew Chan First of all, I do not think you need to treat your double digest with CIP (you used two different enzymes). As Pierre suggested, you vector is not fully digested!
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