I want to clone a gene in Pgemt vector having BamH1 frw enzyme and Sal1 frw enzyme. where as in Pgemt vector BamH1 is considered to be a enzyme which "Restriction Enzymes That Do Not Cut the pGEM®-T ". I am getting positive colonies and confirmed it by colony PCR. But when doing digestion it is not possible...
Hi. At first i suggest to simulate the colony PCR with a software like "oligo" to be sure that your primers don't have any site on pGEM. also you can use other restriction enzymes that have recognition site on your fragment or on the plasmid then check fragment sizes.
Hi there,
If there is no BamHI site in the vector, why are you using this enzyme for cloning? There is no chance to get the right construction...
hi !!
You can choose a restriction enzyme that has one restriction site in the insert and one in the vector. u can digest the plasmids that showed positive in colony pcr with this enzyme, run it on gel and check which colonies give your desired bands.
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