Hello! I'm trying to perform site-directed mutagenesis, similar to the QuickChange II protocol, on a plasmid of approximately 9 kB using Q5® High-Fidelity DNA Polymerase. I've attempted various reaction conditions, but none of them appear to be effective when I examine the product using agarose electrophoresis. Here are the details of the reaction:
- Forward Primer (fw): 47.1% GC - Tm: 65.8°C
- Reverse Primer (rv): 47.1% GC - Tm: 64.4°C (Both primers are targeted at the same region with one mismatch corresponding to the desired mutation)
- Template DNA: 10 ng
- dNTPs: 10 micromoles
Initial denaturation: 98°C - 30s
Cycles (25): 98°C - 10s 65.1°C - 30s 72°C - 10 min
Final extension: 72°C - 2 min
Any suggestions? Thanks!
From the Quikchange II manual: "For long (~8 kb) or difficult targets, we offer the QuikChange II XL Site Directed Mutagenesis Kits (Catalog #200521 and #200522)."
Although I agree that Q5 should work fine on a 9.5 kb template, I've found that using different polymerases with the Quikchange kit doesn't work well for some reason (although I haven't tried Q5). If you can afford it, I would buy the XL kit and follow the manufacturer's instructions.
I got very good at site directed mutagenesis using partially overlapping mutagenic primers using this method: Liu, H., Naismith, J.H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol 8, 91 (2008). https://doi.org/10.1186/1472-6750-8-91
What works for me is 3 - 6% DMSO, 200 nM primers, 200 uM dNTPs, standard amount of magnesium, 100 - 200 nanograms of plasmid template DNA.
Initial denaturation: 98 celsius (3 minutes)
Cycles (x35):
Melting: 96 celsius annealing (30 seconds)
Annealing: 60 or 65 celsius (30 seconds)
Extension: 72 celsius (4 minutes)
End cycles:
Final extension: 72 celsius (4 minutes)
Phusion DNA polymerase takes just 4 minutes to go around a 8 kB plasmid.
But if Q5 isn't like Phusion, maybe do a longer extension time and final extension for the same time.
Don't worry about using so much template DNA. You can remove it from the PCR product reaction by adding DPNI enzyme after.
Hi,
I agree with Adron - you can use a little more template to increase yields.
As for extension times, I usually add a bit more time (more than 1 min per kb) when dealing with templates longer than 8 kb, as polymerization can take a bit more time for longer DNA strands.
Also, 22 cycles should be more than sufficient for maximum yield. At 18-20 cycles, you are likely to be in plateau.
Good luck!
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