Hi I hope someone can help. I am trying to clone a murine TCR, and to do this i have introduced a ClaI and BglII site into the PCR product. I have done this and cloned it into pGEMT, and sequenced it to check that the sites are there, they are. Also there is a unique BamHI site in the insert as well. Here is the problem. I try to cut the vector with the above three enzymes and also XmnI (unique to pGEMT) and I get what looks like partial cutting (but i dont think it is) with all four enzymes. If however i cut a control plasmid with the XmnI enzyme without the insert the cut is complete.
1. I have tried to purify the mini preps so they are clean.
2. I didnt overload the system with DNA so that some would be uncut.
3. Sequence looks fine.
Q: Why does the pGEMT specific site cut just as bad as the others when a control plasmid prepared the same way cuts well???
Q: Can the DNA insert create some weird secondary structure that makes the plasmid hard to cut??
I urgently need help with this because i have been held up at this point for over 12 months. I have tried so many things, so why isn't it working??
Thanks for your advice guys.