Hi folks! I want to subclone the S. pyogenes nickase (D10A) gene into certain expression vector by PCR-amplifying it from a Cas9 template and assemble this with a regular isothermal enzymatic reaction. As the D10A mutation is located 29 bp from the start codon, the wanted changes were included in the fwd primer, 3 nt from the 3´ end. After sequencing some clones, the insert is only the wild type variant. I though that -very much against the odds of geometric amplification- with too much template, I might be picking clones that came from heteroduplex inserts in which the 5´exonuclease of the assembly reaction was sweeping out the desired mutation. So I did an asymmetric PCR to enrich for the watson strand, then the full PCR, but the clones are wrong still. Am I missing something fundamental here? I will appreciate your comments. bye!
Most of these approaches nvolve making changes in either the 5' end of either primer, not the 3' terminal ends as you've done. The other option is to make the mutation the middle of a long primer and amplify the whole plasmid (pfu style). A priori your approach may work, but it is problematic due to possible template switching. Also, if you use a Gibson type assembly (iosthermal enzymatic is rather vague), you need homology regions. If you are using classic T4 ligases approaches, make sure you kinase your oligos. Do you see a clear band of the expected size after PCR? If not, your PCR may just fail.
Good luck!
A somewhat dated approach but one that has worked well in my lab is described in Michael, SF.1994. Biotechniques 16(3):410-412. Our experience is that picking 3 transformants for sequencing give 2 out of 3 desired mutation. Good luck!
Are you sure these are even assemblies at all? It sounds to me like you're simply re-transforming the original template plasmid, and not getting any assembly at all. Can you tell? Is the template backbone different from your destination backbone?
If I'm right, you would fix that by diluting the template plasmid to the point where the PCR just barely still works. Could be 1:500, then 1 ul into a 50 ul PCR reaction. At that point you would have eliminated template carryover and rare assembled clones might start showing up.
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