how to determine pad using Kozich template

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?



First of all, we don’t really have any evidence that the 2nt pad helps or doesn’t help :slight_smile: 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.


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 :slight_smile: