Can someone help with my restriction digestion problem with pGEM T Easy vector?
I am trying to produce recombinant GH, so the first step was to amplify by PCR the gene, besides the sequence of the gene, the primers also contain restriction sites for EcoRI 5' and NotI 5'. After the PCR, the fragment was purified by gel extraction, then the fragment was ligated with pGEM T-easy, I grew up colonies with the product ligated, then I made a miniprep. But when I want to cut the pGEM-T easy with EcoRI and NotI, the restriction digest is not produced, I expect 2 bands on the gel, one of 650 pb (the insert) and the other of 3000 pb. At the same time I have another vector (pCAG with GFP, I want to use this plasmid to produce my desired protein) that I digested with the same enzymes so I can remove the insert of GFP (600 pb), but in this case the digestion worked. Another information to add is that the concentration of both plasmids for the digestion was 3 micrograms and I added 2 microlitrers of each enzyme. I don't know if it has to do with the concentration of the plasmid or the enzymes.
Edited by liligompac, 25 April 2018 - 09:15 PM.