I ran a PCR reaction and it gave good result during the trial run. However, once I ran the same PCR reaction on all of the other samples, there are smears and appearance of non-specific bands. I'm not sure on what went wrong. Hopefully, I could get some insights to fix this issue. Thank you in advance!
you have a lot of primer dimer so use less primer and ideally use a hot start enzyme. If the 2 larger bands persist then run a gradient of annealing temperatures to find the best temperture for your primers to anneal to the template dna
Paul Rutland rightly said! On top of that you can also play around with the MgCl2 concentration to fine tune your PCR. I say this because I do see some amplification there. To reduce non-specific amplification, reduce the concentration of MgCl2. Most importantly, get the annealing temperature, primer concentration and MgCl2 concentration right. This generally solves the problem.
Nurin Jazlina It would be of great help if you could specify the mixtures that were used in those amplifications.
I'd look into how u store your target DNA samples first. Are they the same samples that had been used on the trial runs? (look for contaminations in containers/ storage space, etc.)
If it's not the samples then I'd look into the mixture and working environment.
Need to cross out the "human factor" first!
Primers can degrade over time from repeated freeze-thaw cycles. If nothing else changed (e.g. same thermocycler settings, same concentrations of reagents), try making a new dilution for each primer from the frozen stock solution. Or just order a fresh set of primers. Primers are cheap, your time is valuable.
Good luck!
Paul Rutland Swapnil Sahoo thank you for the recommendations! I have performed the gradient PCR, as well as reducing the primer concentration and my gel is showing crisp bands with no appearance of primer dimer. However, genomic DNA was the one being amplified with appearance of non-specific bands, instead of the target region. I will be focusing on troubleshooting this issue and any advice would be of help :)
Katie A S Burnette I made a new batch of diluted primers for my PCR reaction and there are no appearance of primer dimer in my PCR product. Thank you for the advice! :)
Anh Trần Nguyễn Bảo thanks for sharing your thoughts! I'm storing my extracted samples and primers in -20C freezer. Yes, I did used the same sample for my trial runs and recently, I had made some adjustments on the annealing temperature as well as the primer concentration. Its giving crisp bands except its not amplifying the target regions.
I think we need to act on the concentration and quantity of MgCl2. if you had used the same taq polymerase as during the calibration of the PCR reaction, adjust the hybridization temperature.
Nurin Jazlina I think that the non specific bands are not an issue as they are much too big to confuse with the correct amplimer but some ideas for getting rid of them are to reduce the annealing and extension times in the pcr and BLAST the primers to make sure that neither primer is in a repeat sequence. Some repeat sequences are very common and can also be inverted so you could try running a pcr with just one primer ( F and R in different tubes) which should fail but works if the primer is not gene specific. It still worries me that the control works clean and well so I would check the OD260/280 of your control and your samples and run dilutions of your dna in case of pcr inhibitors and use more MgCl2 in the reaction but while troubleshooting try to get your samples at exactly the same amount as your control sample. Katie A S Burnette suggestion of designing new primers is a good suggestion both for amplification and also for reducing non specific amplification
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