I am in pWe15 by stratagene with a 45kb insert. I cannot make any of the primers work. I have tried up to 100 cycles. I have added 5-10% dmso or 5-10% gc melt.
my denaturaation is done 1x2min 98c and 100x 30sec 98c. What am I not doing??? what can I do differently???
your help is greatly appreciated
David S. Fruhling
Deptment of Oncology
Wyeth Ayerst Research
fruhlid at war.wyeth.com