I was doing pathway engineering which involved joining of 6 DNA fragments. I have joined fragments 1 and 2 and named it as A. I have joined fragment 3 to 6 and names it as B. Now when I am joining A and B, I am getting smear on the upper portion of the gel where my band of desired size should be. I am using Q5 high fidelity DNA polymerase from NEB.
Agreed with lengthening the elongation time. Especially if it's a tough template, the minimum recommended time is usually not enough. I'd try doubling the elongation time from what you've been using. Maybe increase the annealing time as well. In addition, you could try adding some DMSO (0.5% of total reaction) or try using the GC enhancer if that came with your Q5. Those might reduce any secondary structure problems that may be occurring.
Are you using the forward primer of A and the reverse primer of B to amplify the final product? If that's the case, it might be that those two primers don't work well together. A lot of times when designing things like this people put a lot of design effort into the primer pairs for fragments A and fragments B but don't consider that the the forward primer from A and reverse primer from B also need to work with each other and not form excessive primer dimers, etc. I've definitely been guilty of this and ended up with smears or multiple bands that I had to gel extract and hope for the best.
Agreed with lengthening the elongation time. Especially if it's a tough template, the minimum recommended time is usually not enough. I'd try doubling the elongation time from what you've been using. Maybe increase the annealing time as well. In addition, you could try adding some DMSO (0.5% of total reaction) or try using the GC enhancer if that came with your Q5. Those might reduce any secondary structure problems that may be occurring.
Are you using the forward primer of A and the reverse primer of B to amplify the final product? If that's the case, it might be that those two primers don't work well together. A lot of times when designing things like this people put a lot of design effort into the primer pairs for fragments A and fragments B but don't consider that the the forward primer from A and reverse primer from B also need to work with each other and not form excessive primer dimers, etc. I've definitely been guilty of this and ended up with smears or multiple bands that I had to gel extract and hope for the best.
Check your primers compatibility with each other. You can increase extension time and annealing temperature. MgCl concentration is also a big matter in PCR. What is size of desire band?
Hello all,
The desired band size is of 4 kb. The primers are compatible with each other since their Tm falls between 5 degree. I do add GC enhancer which works with Q5 polymerase. I will try increasing the annealing temperature. Regarding elongation time, I used 2 min because Q5 polymerase has a rate of 20-30 sec per kb.
if there is sequence homology between parts of a and B you may be getting annealing where you do not expect it and increasingly longer amplimers. Firstly try increasing your extension time to 4 minutes in case some of the smear is single stranded and incomplete ds DNA in spite of what the manufacturers say works
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