I have been using a pair of primers (designed by my co-supervisor, who is no longer here) to amplify my sequence of interest from chelex extracted genomic DNA. Even though I always had a faint band below 250bp in my samples, positive control and negative control (primer dimer?), I always had relatively strong amplification of the sequence of interest.
BUT, after a few months I started to ONLY have a very, very intense band below 250bp (in both my samples and controls) and no amplification of my sequence.
I tried changing the parameters of the PCR (HotStart, different annealing temperatures, dNTPs conc., primer conc., enzyme conc.).
Also, there should be nothing wrong with my genomic sample, since I used it to amplify another sequence with different primers and it worked perfectly.
So I don't know what else to try.
Could my primers have stopped working all of the sudden?
I think that if your negative control has strong bands of any size then you have post pcr contamination, This will amplify very well and take over the reaction , Change all of your reagents including the water and thoroughly clean your pipettes inside and out, Clean your working area, When the new primers arrive dilute working stock tubes with another workers pipettes in another working area and set up the new pcr away from your working area, If this works then move back to your working area and pipettes to see how successful your clean p procedures have been. Small amplimers always amplify better than large ones so any contamination ( amplimer or primer dimer) will always take over the reaction and cause low amplification of the real target sequence
Thank you very much for your answer.
I will try with new reagents and with other people's pipettes (I had not thought of that last thing)
In addition to considering the contamination, I believes that primer degradation may also be one of the reasons. If the primer is not placed in an appropriate storage environment, the storage time will not be too long, especially the repeated use of primers. I usually dispense the newly synthesized primers and try to make sure that the primers and ddH2O used in the lab are new each time. Primers that are not used are stored at -20 degrees centigrade. If you are unfortunate enough to encounter primer degradation, re-synthesize the primer as soon as possible. Good luck with the experiment.
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