Hi, I have a question regarding my PCR products. Okay so i did some primer optimization for primer 1 using gradient temperature starting 55-60’C and i chose temperature 56.6’C as my temperature as it show a clear band. However, when i did amplification using primer 1 at 65.5’C for all my 60 samples, they showed double bands which are quite questionable as they did not even showed up when i did my optimization. Can someone help me to identify the problem? I did the same step with the same primer and the same machine. Here, i attached my result. For your information, 1e is the band for 56.6’C.
I think that you have run the second gel too fast and also that yu have added too much primer to the second PCR. The ladder has run a long way through the gel. It looks like the smaller band is a primer dimer. If you run a no template control (water instead of DNA) and you get the smaller band then it is primer dimer. This can happen with too much primer or by leaving the pcr mix for too long before amplifying or by having too much magnesium but too much primer seems likely to me
I used the same amount of primer and to be clear, during optimization, i used 80V and during amplification, i used 70V to get a clearer band of the marker. Maybe i took a lot of time before amplification. But how do i fixed this problem? I have like 60 samples to run at the same time. It is quite time consuming. Anyway, thank you for your time in answering my question.
If it does turn out to be primer dimer some options are;
1 Amplify smaller numbers in each assay.
2 Prepare the plate of samples on ice.
3 Prepare the 60 samples with a mastermix without Taq then add the diluted taq when all the samples are ready to run. Work on ice ideally .
4 Use a hot start enzyme.
When running agarose gels do not go above 5v/cm where cm is the distance between the electrodes of the gel apparatuse so 20cm between electrodes would be 100v
If the smaller bands are primer dimers, you can also reduce the concentration of primer pairs in the reaction mix or check the primers with a primer design software.
I lowered the primer pairs concentration but it still have the double band
It is still useful to know if the NTC is amplifying but if we treat the 2 bands as real amplimers then I suggest running a dimethyl sulphoxide DMSO gradien. Set up 7 identical samples but in each tube the dmso concentration (final) should be 0%, 1%, 2%,4%,5%, 6%, 7% and 8% dmso. At some dmso concentration the lower band should disappear
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