I recently did a PCR with certain primers, and the reaction went well for two gels, but when I repeated the exact same reaction a third time it suddenly didnt work, then I did it a 4th time and it worked but the gel band was extremely dim, so my question is why?
it was not evaluated well in the beginning of the pandemic
now we are seeing things from a different perspectives
One common reason is repeated freeze-thaw of PCR reagents, especially the primers. Changing all the reagents might solve the problem. If the same samples (DNA/RNA/cDNA) were used again and again that might also get worse, if possible, running a gel of the samples would be a nice way to be confident that the sample is ok.
I totally agree with Sayanta, additionally, I would like to add:
Sometimes, the primers have a tendency to form loops or dimers amongst them, so one thing that you could try is, heat your primers for 7-10 mins on a heat block then keep on ice and add immediately to the PCR cocktail. This way the number of primers available for PCR is more, hence, more product formation.
Agree with Sayanta,
A nice way to do this (IMHO) is to take aliquots of your primers, enzymes etc.. Then you will only freeze-thaw your regents once or twice.
Check out https://www.thermofisher.com/ca/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html#:~:text=Check%20amplification%20length%20capability%20of,primer%20binding%20and%20enzyme%20thermostability.
I was facing the same problem last year, the possible reasons are (I also tried them all one by one):
*First of all check if your samples are still usable.
1- Contamination of the primers- primers usually get contaminated due to mishandling and pipetting errors. Also do not add primers in PCR reaction mixture without completely thawing it.
2- Primers are old- Also if the primers are not new, there is chance of them getting contaminated more easily. Humidity in primers (prior to using them) also may effect the PCR results.
3- Making aliquots of samples as well as primers- It will reduce the chances of degrading their components by repeated freeze-thaw process.
4- Nanodrop - or quantify in any way possible your total extracted DNA/RNA (whatever you're working on) before doing the PCR.
5- Problem with the agarose gel- There might be some problem with the gel you're running like change in voltage, agarose concentration and quality, TBE buffer. Trying making the new buffers and whole gel from scratch so the chances of contamination are minimum.
6- PCR kit reagents or mastermix- There might be a problem with the reagents you're using.
7- Run a negative control- To check for contamination either in your kit reagents, primers and sample running a negative control is best option. To run a negative control do prepare the PCR mixture in your normal routine way BUT do not add the the RNA/DNA sample you're working on. If you get a clear gel of negative control PCR product then it means there is no contamination in kit reagents and primers.
8- Do a gel of total extracted DNA/RNA- If you don't have nanodrop, try doing this to get an idea if there is a problem with extraction.
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