saturation mutagenesis library problem again - (Sep/09/2011 )
I tried to saturate another single amino acid position, but the result showed almost abundant with wide-type nucleotide again (in the attachment).
So I am wondering what can I do for this situation?
Thanks in advance!
-Biogareth-
I would say that is entirely your wild type, but perhaps the baseline is more convincing in other parts of the sequence. How are you doing the cloning and elimination of the template? Is this done with Stratagene Quikchange kits? Usual problems: too much initial template, failure of the DpnI digestion, failure of the PCR (leading to selecting only the wild type). Tell us a lot more about EXACTLY how you are doing this.
-phage434-
phage434 on Fri Sep 9 11:31:53 2011 said:
I would say that is entirely your wild type, but perhaps the baseline is more convincing in other parts of the sequence. How are you doing the cloning and elimination of the template? Is this done with Stratagene Quikchange kits? Usual problems: too much initial template, failure of the DpnI digestion, failure of the PCR (leading to selecting only the wild type). Tell us a lot more about EXACTLY how you are doing this.
Hi Phage434,
You are right it looks wild type. The procedure I used actully the same like before. The procedure works well for several times. But it is not always successful.
The elimination of PCR products should be Okay, because I also check DpnI-treated mixture by SDS-page (in attachment, plasmid size 5.2kb, megaprimer size is 1.7 kb).
-Biogareth-