I had to insert a 1.5Kb piece into 2 different expression vectors pEGFP-N1 and pHM6. I PCR'd the insert from a clone I bought from ATCC with the primers for the restriction sites for the 2 vectors (EcoR I and BamH1 for pEGFP and Eco RI and Not 1 for pHM6).
These primers also had the the kozak sequence on their forward primers after the restriction sites. The 5' end of the primers had an 'ATT' sequence added. So, it will be ATT-restriction site-kozak-primer in that order for the forward primers and ATT-restriction site-primer for the reverse primers.
I have been getting the correct size bands in each step and also the colonies, but there is no insert in them. Please advise me and let me know if these primers are OK. Also, I heard about blocking of cleavage by CpG methylation in Not 1 enzyme. Has anyone encountered this kind of blockage and what is the solution?
I have also tried TOPO TA cloning with the same primers without the ATT and the restriction sites, but have not got any results!
Edited by jummu, 30 June 2004 - 07:16 PM.