I need to do gateway cloning but I currently have some problems amplifying the cDNA of my gene. I suspect that my primers might be the reason but I´m not sure! The problem seems to be that the forward primer starting at the ATG has a Tm of about 55°C (I varied primer lenghts but all have similar Tm´s) whereas the reverse primer at the stop codon always ends up with a Tm at around 73°C (+/- 4°C).
I already tried different taqs, templates etc. without success. Does anybody have a suggestion/idea how to solve the problem?
Thanks for help and all the best
Maybe I should mention - I used the Tm calculator from finnzymes because we´re using the Phusion taq for cloning!
Edited by nimbus, 25 February 2010 - 11:46 AM.