Hi Angie,

I would recommend a couple of things. Perhaps the most obvious thing to do is get the primers remade--especially if they are old and have been thawed multiple times. Less likely is that there's something going on with your PCR machine. I have seen results similar to yours when a PCR machine malfunctions. Try the experiment on a different PCR machine to try to rule that out. Lastly, I would double check your cycling conditions to ensure that someone hasn't accidentally changed them.

If you can rule out the thermocycler, my first thought would be the primers--especially if you feel the DNA is still good.

Hi Angie I want to ask you Is the samples were the same that u used one year ago? May be the genomic DNA was degradated. You can make a gel for genomic DNA.

The 100bp band appeared in negative control if yes so it will be a primer dimer.

I hope my answer will be helpful

if the primers are dissolved in water and not in TE then acid depurination may have degraded the primers. You can try adding 5x the amount of primer in the hope that some remains but best to get the primers remade

Hi, Angie,

I agree to all recommendations above. Obviously, if you have been using the same primer pair that you have used last year, you have to consider how you stored and prepared the primers. Primers dissolved in water easily degenerate compared to those in TE. Multiple thawing-freezing cycles might have also affected the integrity of your primers. Consider ordering a new batch. For your polymerase, check if it's of the same optimal temperature with the one you used before, this might have caused the problem, too.

Hope this helps.

Best,

Arizaldo

For the last time that I did the PCR, I have used a new primer stock but still did not see any bands. This stock was also ordered last year with the same sequence but stored at -20 degrees and have not been freeze-thawed. The polymerase was from the same batch as last year but was from a fresh vial that has not been freeze-thawed. I have also checked the cycling conditions before the experiment.

The samples were prepared last month and I have used them for PCR and sequencing of other genes successfully. Hence, I am puzzled as to why the PCR does not work for only this primer pair. I do not have access to other PCR machines.

If there is a problem with the primers, would it still have worked for some reactions and not others? I guess I will order fresh primers and try again.

Thank you for your suggestions!

Hi, Angie,

Aside from ordering a new batch of primers, you may look into the possibility of uneven heat distribution in the wells of your thermocycler.

Take a look into possible PCR inhibitors. You may refer to the following article: https://www.promega.es/-/media/files/resources/profiles-in-dna/1001/an-introduction-to-pcr-inhibitors.pdf?la=es-es

Hope this helps.

Best,

Arizaldo

in answer to the question why would poor primers work on some samples pcr needs a huge excess of primer to anneal quickly and amplify, If the primer concentration drops and some of your samples have inhibitors still present while others are very pure it might be that the pcr inhibitors have more effect on low primer concentration so these are the samples that do not work as well and even a 2 per cent drop in efficiency would mean only 54 per cent yield which might over 30 cycles be invisible on a weak amplification

Use fresh compounds. Sometimes the buffers are interchanged (TE not TBE!). Use a control DNA with control primers. Check the cycler. Do not use distilled water. How did you check the presence of amplified DNA? Is the DNA size standard clearly visible in the gel? etc. ...