I used to do this kind of thing and now have forgotten strategy please help and be specific. The vector is now linear having been digested with EcoRI and MfeI, then treated with CIAP, to prevent self ligation, and gel purified.
I attached the vector map.
The EcoRI and MfeI digested CIAP treated gel purified vector sequence is 4585bp
I am at a point where I have the 3049 insert fragment with EcoRI and MfeI ends. This should be able to self ligate. If it does, there will be nothing but my gene of interest and that will not support bacteria in the presence of the selection drug Kanamycin.
I remember ratios of insert to vector being useful parameters to fiddle around with. The idea was that only the right stuff would live in the presence of selective agent.
Please suggest ligation strategies and do not be concerned about being overly pedantic. I would prefer an overly pedantic approach.
This should be standard cloning as the insert size is not too big. So if you do Vector:Insert ratio of 1:3 it should work. It is good that you depphosphorylated the vector which should reduce the background. This calculator is very helpful to calculate the exact amount of Vector/insert to be sued in the ligation reaction.
http://www.insilico.uni-duesseldorf.de/Lig_Input.html
For eg, if you use 25ng of vector, then add 50 ng of insert DNA to achieve the 1:3 ratio.
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