Because I have succeeded in some other positions with NNK primer, I compared those good primers with this bad primer, and found that the stability of the primer looks quite important, therefore, I redesigned this primer by replacing three nucleotides on both flanks (silent mutation) in order to get the higher stability.
The primer is as follows, and the replaced nucleotides were shown in red:
Do you think is it reasonable to design the primer like this?
Any suggestion is welcome!
Edited by Biogareth, 13 September 2011 - 02:40 AM.