I am attempting a deletion from purified plasmid using the NEB Q5 mutagenesis kit. After mutagenic PCR I ran a gel to attempt to observe the band corresponding to the length of my mutagenized plasmid. The expected band was present, but so was a "smear" below it. I'm assuming the smear consists of incomplete PCR products that were not amplified to match the full length of the plasmid. Based on the way the Q5 kit works, with primers oriented back-to-back and polymerizing away from each-other, I'm thinking these incomplete products will be incapable of circularization (lacking either compatible sticky ends or fully blunt ends) in the KLD reaction and are thus a non-issue. Is my thinking correct here? Anyone else have this experience? Thanks!
I have been doing my own KLD based SDM using self-assembled enzyme mix, i.e.: Q5 polymerase, and KLD steps with Promega kinase + ligase and NEB DpnI in Promega ligase buffer. As I worried about the efficiency of DpnI, I purified the PCR products by gel purification to ensure total removal of the parental plasmid. (this is optional)
The KLD mix will ligate blunt ends. So your "incomplete products" could ligate.
I usually have no smearing on by Q5 PCR products. Have you also check on specificity of your primers using primer blast? I suggest u do gel purification, before KLD.
And how long is the expected products? If those are incomplete products, you might want to increase the extension incubation time in amplification cycles as well as final extension steps.
I agree with the previous answer, that your incomplete products could still possibly ligate. If the problem persists I would suggest just doing a gel purification from the correct band. It's not the most favorable course of action, but if you have a high volume of product you should be fine.
Hey all - sorry for the long delay, I appreciate all your responses. I went ahead and did the KLD reaction as described in the kit protocol, and have now confirmed by sequencing and restriction digest that I got the deletion I was looking for (Hooray!).
My logic for why the smaller bands ended up not mattering is that the only possible non-mutated double-stranded products resulting from incomplete PCR would be either A) only overlapping in the middle, with long non-complimentary single stranded sticky ends or B) blunt, but hemimethylated and thus degraded by Dpn1. This is resultant of the primers sitting back-to-back and polymerizing away from each other, only "meeting in the middle" at the opposite pole of the circular plasmid.
If anyone else encounters this issue I'd say go ahead and do the KLD, but be aware that you probably have less of your desired product present in the PCR than normal. You may benefit from using slightly more of the KLD product in the subsequent transformation.
Cheers and thanks again for the input!
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