Mystery in my PCR
Posted 09 July 2013 - 06:18 AM
Please help me figure out what happened with my PCR.
I used the same master mix, same DNA template, same condition of thermocycles for 4 reaction tubes (10ul/tubes). However, I just only got the band in 1 tube when I checked on agarose gel. I repeated the experiment, but got nothing. I tried one more with 2 tubes and new aliquoted primers got from stocks, and I got products in both. So, I tried again with 4 tubes (because I need high quantity of PCR products), and got nothing...
I dont know what was the problem. I repeated several times, but the PCR just worked in some times and some reaction tubes...
I am so thankful if you give me some suggestion...
Posted 09 July 2013 - 07:27 AM
Posted 09 July 2013 - 05:14 PM
At first, I used gradient PCR to optimize the annealing temperature (from 60-68oC) and used Pfu DNA Pol. I chose 63oC to use as the annealing temp for my experiment, others also showed the bands but of course not specific binding. Thus, I don't think the annealing temperature was the problem.
Next, the primers did work because whenever I tested with gradient PCR or test with 1~2 tubes, I got the bands. I wondered about the secondary structure of Primer or primer dimer, so I used the tool in idtdna.com the analyze my primer, and it showed 2 hairpin structure formed at 47 and 48oC.
I still worry about the primer, maybe it work unstably... How can I check the primer if they can work well or not?
Thank you again for suggestions.
Posted 09 July 2013 - 05:34 PM
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Posted 10 July 2013 - 07:09 PM
Posted 11 July 2013 - 10:43 AM
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