Dear
I prepared my cloning reactions with positive and negative controls of my reaction. The positive control was pET with double digestion in presence of T4 DNA ligase and the negative Control was double digested pET without T4 DNA ligase. I electrophysed the reactions by agarose gel. I can see the DNA fragments in negative control on the gel but I can not see the related bonds after incubation for ligation reaction and positive control. I want to know is it logical and how we explain this? Thanks
The double-digested vector can ligate into even-numbered concatamers (2-mer, 4-mer, etc) that in turn, may be circularized or not, so you end up with a population of multiple sizes and relative mobilities where each species is not abundant enough to produce a detectable band on EtBr-stained gels. Same (or even worse) for the vector+insert reaction.
Also -according to my colleages, I'm not into checking ligation reactions by electrophoresis- you might want to inactivate the ligase by heating to 70*C for 5 min before loading onto the gel. DNA-binding proteins tend to alter electrophoretic migration (this is commonly experienced in the lab with restriction enzyme digestions).
Hope it helps!
Yes, what you see is an expected result.
Normally, the recommended conditions for ligation include considerable excess of the enzyme and you will see large polymers. If you really wish to a single event, used far less ligase.
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