Questions about Stratagene QickChange Multi-mutagenesis - about how many primers and template DNA size (Jan/31/2008 )
I found one of my plasmids contains several unexpected point mutations and wanted to mutate them back to the wt sequence. The problem is the these mutations are well separate and one primer cannot handle all the mutations. So I plan to use Stratagene Multi-mutagenesis kit to do the work. I will need 8 primers for the mutagenesis; the size of my plasmid is ~ 10.8 kb. After read the kit instruction, it seem to me the kit can handle at most 4-5 primer in one run and the template DNA size should be below 8 kb.
Can I use all my 8 primers in one run?
My plasmid is ~10.8 kb, bigger than the suggested 8kb at most. Is the mutagenesis reaction going to work for my plasmid?
Any suggestions will be greatly appreciated!
I haven't used this kit, but do you need to change your point mutations? Maybe they are silent codon changes that won't do any harm in most cases?
Otherwise, maybe you can subclone parts of your vector, do the mutagenesis (multi or single) and clone back (will be a lot of work though).
Other than that: I believe the reaction mix of the kit to contain Pfuturbo and PfuLigase (Turbo for DNA polymerisation and ligase to connect to the primers along the way) so maybe if you combine the improved PfuUltraIIHS (capable of longer fragments and higher fidelity) with PfuLigase in the quickchange multi-buffer (or just PCR buffer, not sure if the ligase needs much more than ATP in a pH-buffered solution) you might be succesfull for such long template?
Thanks for advice, Vairus.
You are right that there are a few silent mutations, but I still need to change at least some of those mutations back. I found 11 nucleotide mutations and 4 points mutations in amino acid level. I will try the kits first, if not successful I would try the reaction mix of Pfuturbo and Pfuligase. Thanks for suggestions.
Might it be easier to reclone? Are you sure the wt sequence is correct? The clone may be from a different strain and just as viable.
The wt sequence has been published. I got the DNA from the author, which was supposed to be wt. I think maybe those random mutations were introduced during the bacteria amplification.
They could be sequencing errors. How much do you trust the literature? How old is the sequencing data? Old sequencing (pre- 1993 or so) is pretty unreliable.
If this is an E. coli gene, you can just PCR it from the genome.