BSP PCR primer design explained - (Feb/11/2013 )
pcrman and phage434, thank you so much for all of this input. I think I have a better grasp of the primer designing issue now. I see why this step is the most essential (and complicated!)
vilperte, thank you for that website! I tried it out and I think it's much easier to understand than the other primer designing programs out there. It would be great to hear about the polymerase you're using and how it turns out.
I just have one more question...I really do understand why the amplicons should be much shorter than what I have, but do you think it's impossible to get results with a larger amplicon? I'm hesitant to abandon my current experimental design for larger amplicons, but if it will never work, I will move on.
When you do the bisulfite conversion, it degrades a huge amount of your DNA, so your DNA will be very fragmented.
It's not impossible to to get results from large fragments, I've read some comments from people that managed to amplify some fragments up to 1kb.
But it is very unlikely that you'll have an intact DNA with great amounts in that region.