I’m trying to design a couple of sets of primers for ciliates based on your template. I’ve got a handle on the linker (adjust melting temp) but is there a systematic way to determine what the pad should be?
thanks
I’m trying to design a couple of sets of primers for ciliates based on your template. I’ve got a handle on the linker (adjust melting temp) but is there a systematic way to determine what the pad should be?
thanks
Hi,
First of all, we don’t really have any evidence that the 2nt pad helps or doesn’t help So, you could always just ignore it if this proves impossible. I think what I did was to trim everything to the left / right of the primer and then look to see what bases were present in those two positions. I then picked the two bases that didn’t show up or were the most rare. You’re basically looking for a pad that is not complementary to what is found in real life.
Pat
I ended up just checking a few of the indexed primers for hair pins and went with that. We’ll see if it works. Hopefully in a few months there’ll be a fab methods paper about miseq and ciliates