My PCR amplification of a 480 bp fragment for human genomic DNA is not working. The primers were designed to anneal to a region flanking the BRAF gene. It used to work before and now I only get primer dimers in my gel. The reagents such as Taq are not the issue because it amplifies another 369 bp fragment using another set of primers. I tried using a fresh aliquot because I suspected my primers may be degraded. But I still only get primer dimers. Please help me out with solving this issue
Did you check out the primers Tm and the presence of hairpin in them ? Also what are the parameters of your PCR cycle ?
I checked the Tm and I've performed a gradient pcr. I optimized the conditions and I was getting a good product. It suddenly stopped working. Now I only get primer dimers. My primer length is 44bp because I had to match it to the Tm of the other primers. I didn't check hairpin formation though. My parameters: 95 for 5min, 94 for 30sec, 62 for 30sec, 72 for 30sec, 72 for 6min, 45 cycles
Following on Mikael's suggestion: too much genomic DNA can inhibit PCR. Make sure you use absolutely tiny amounts of it.
One time my previously effective PCR suddenly stopped working was when I used a cycling program that had shortened the initial denaturation step from 10 minutes to 3 minutes. This step is often required not only to unwind the DNA double helix, but also to activate the enzyme, so shortening it can actually be detrimental.
Consider everything that has changed since the time the PCR was working. Do you have a new batch of primers? A new batch of the PCR kit? Are you using a different cycler? If possible, try to reproduce the method when it was working exactly.
First, you should do the following experiment:
Pick a set of samples (5 - 10).
Set up PCR in duplicate or triplicate with your problem primers and the primers which give you the 369 bp product. Use exactly the same reagents (except the primers). If possible, run the samples on the same instrument in parallel.
Compare the results. If all the 369 bp PCRs are positive and all the 480 bp samples are negative, then you have a problem. It might be the primers; primers are cheap so ordering some fresh ones makes sense. It might also be an instrument problem. This can easily knock out one PCR, while leaving another, more robust, one working fine. Check your instrument calibration if you have access to the required test instrument.
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