I have amplified this 5 kb fragment using LongAmp Taq DNA polymerase from genomic DNA, then I extracted the amplified product from the agarose gel and I have purified it. I have tried amplifying this purified PCR product using Q5 polymerase, which is the only enzyme I can use for PCR now, to no avail. I have calculated the annealing temperature and the elongation time (3 min and 35 secs) according to the Q5 protocol.
I've seen other people suggest to scale down the annealing temperature by 2 C degrees. Some other people also suggested to double up the elongation time to 6+ mins. What would you suggest me to do?
How much template do you use?
If your template is GC rich you can use the high GC enhancer that comes with the Q5 enzyme.
you can use gradient PCR to optimize your annealing temp.
You might also trry different primer concentrations, from my experience the less the better. Regarding annealing temperature, I usually do gradient from 52, 54, 56, 58, 60. For elongation, I do one minute/ 1kb, so 5 minutes for you. I would also recommend to Always alter one variable at a time.
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