I'm trying to linearise a plasmid that I will use as my vector for NEBuilder HiFi DNA Assembly. I have designed primers for linearising the plasmid that I am now testing.
There are shorter bands appearing in my gel from the PCR products that shouldn't be there and I think it's because of the PCR conditions I've been using and am wondering how to optimise it.
I did a gradient PCR which showed my desired products at an annealing temperature setting of 70°C (which was higher than I expected). I had designed my primers to work at around 65°C.
50μl Reaction for PCR using NEB Q5 HiFi dNA Polymerase
10μl 5x Q5 buffer
1μl 10mM dntps
2.5μl 10μM Fwd Primer
2.5μl 10μM Rev Primer
2μl plasmid DNA (15ng/μl)
10μl Q5 GC enhancer
21.5μl nuclease-free H2O
0.5μl Q5 polymerase
THERMOCYCLING CONDITIONS: (according to NEB Q5 HiFi DNA Polymerase Protocol)
initial denaturation 98°C (30sec)
35 cycles:
denaturation 98°C (10sec)
annealing 70°C (30sec)
extension 72°C (40sec)
final extension 72°C (2min)
Are there better methods or ways I can change the protocol to amplify the plasmid by PCR?
I haven't used that exact DNA polymerase before, but a 40 second extension time is very short for a 4.2 kb product. Try increasing the extension time to 2 minutes and see if you get your desired larger product.
- Completely agree with Katie
- For standard Taq you require 1 minute per kb and for old style proof reading polymerases (not so much the new highly processive polymerases like Phusion) up to 2kb per minute
- Accordingly, and acknowledging Katies suggestion I would try a combined annealing/extension temp of 70C for 5 min (after denaturation)
Katie A Burnette Laurence Stuart Dawkins-Hall Thank you! I will increase the extension time, do you think running a Touchdown PCR would help as well?
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