I successfully re- amplified my band of interest once, using 1:1 dilution and increased annealing temperature by 2 degrees. I tried reproducing the same now, its giving me high molecular weight smear. I tried diluting it upto 1: 10 power 6, the smear has reduced but absolutely no bands. Suggestions are welcome.
Hi, Sharon,
When I had such problems, I've tried to make some modifications:
1) make dilutions of initial PCR-fragment (as I understand, this step didn't help you);
2) try to do more wide temperature gradient (in your reason it's not clear, what were the ranges);
3) purchase special DNA-pol with reduced "smear-forming" (cannot suggest universal, because usually use enzymes from local manufacturer);
4) purchase new oligos with higher melting temperature.
Usually, problems can be solved using first 2 steps in combination with 3rd. 4th - is like emergency:)
Good luck!
Regards,
Evgeny.
Hi Evgeny,
shall try that out, although I did try 1 and 2..
Thank you :)
If you have any of the old primers left, test it. Using the combinations, old-old, old-new, new-new! Mostly of the reasons - either [enzyme], [primer], [Mg], [dNTP] used was too high or the annealing temperature was too low for the primers being used. no.of cycles used is many! it may be also due to the long annealing and extension times.
good luck! :)
Hi shyn, thanks.. it makes sense, as my annealing and extension timings are long.. thanks again
I would dilute the first PCR around 1:500 and use the same old primers
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