Okay. I'm running PCR on several genomic DNA templates. I have performed this reaction (using these primers and amplification conditions) several times in the past with no trouble. The last few times I've run the reactions, the product has only amplified successfully in a few samples, while failing (to amplify the band of interest) in the rest.
The troubling parts:
1. Each time I've run the reaction I've used a mastermix - Each reaction should have exactly the same reagents (excluding the template DNA) and they're run at the same time, and at the same temperature, in the same thermocycler.
2. When I've repeated the PCR, some of the samples that amplified in the previous reaction don't amplify in the second, while some that don't amplify in the first reaction amplify in the second!
I'm understandably bamboozled. If anyone has had this experience before and can provide any insight it would be greatly appreciated.
I've attached a picture of my most recent gel. The ladder is 1 kb Plus DNA ladder. The band at approximately 500bp (in the two samples on the lower half of the gel) is the band of interest.
I have had issues similar in the past which ended up being an issue with the thermocycler itself! the temperature across the tubes was not uniform and as a result, some tubes were a few degrees cooler than they should have been.
I guess that's one thing you could check if you haven't already?
Nathan,
Thanks, that's what I thought as well, but I just spoke to my supervisor and he said the machine was tested and calibrated recently and there were no issues.
I'm wondering whether it might be the tubes? It tends to happen when I've run a large batch and I have recently started using 8-strip tubes instead of individuals for big runs (because handling 60+ individuals per run isn't fun).
I would order new primers and go back to optimising your pcr conditions as it looks like you are just on the edge of something working or not working, Possibly the primers are degrading ( they do if dissolved in water not TE) or concentration or contaminants in the dna if the dna purification method has changed. You could try more magnesium and a lower annealing temperature as a first try
Okay. It looks like I've solved the issue. I replicated the experiment exactly, changing only the tubes I was using (from strip tubes to individual tubes) and this appears to have solved the issue.
Paul, I reconstitute my primers in TE buffer and make aliquots to the dilution I require in RNAse free water. It's possible that they had degraded a bit, but it seems unlikely, as two reactions amplified quite strongly - then again, my explanation does't appear to account for this either.
I suspect that the strip tubes either vary in thickness from the individual tubes (they're not from the same manufacturer) or they do not sit in the thermocycler as tightly. Perhaps a combination of both. Other reactions in our lab don't seem to be affected by the change, but having spent quite a long time optimising the PCR conditions for these particular primers, it wouldn't surprise me if a slight change in temperature caused them to fail. A true test would be to make up one master mix, with one template, and perform the reaction in the same thermocycler, running individual and strip tubes concurrently.
Well done David that is a good result. Perhaps a little more pressure from above ( if your machine has an adjustable lid) would keep good thermal contact with the block.
If I could make a suggestion running your checker gel at lower voltage for longer and with a smaller sample might lessen the smiling effect of the amplified bands but I can see that with random results you will have been delighted with a full set of amplifications
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