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a question about degenerate primer (NNK)


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#1 Biogareth

Biogareth

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Posted 13 September 2011 - 01:54 AM

Recently I made a saturation library at a specific position with NNK primer designed by Strategene software, but when I checked the quality by sequencing (see the picture below), it is not good at all because of almost wild-type nucleotides.
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:
GGTTACGAAGGCCACTACTTTNNKGACTCCGACATCTACAT

Do you think is it reasonable to design the primer like this?
Any suggestion is welcome!
Thanks!

BioGareth

Attached Thumbnails

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Edited by Biogareth, 13 September 2011 - 02:40 AM.





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