My PCR's suddenly stopped working a few weeks back, and I can't figure out what is wrong. Attached is a photo of my gel and you can see the segments of smearing which is much larger than my desired product. My target is about 200bp (2 marks up from the bottom of the 100bp ladder). These smears appear around 1500bp.
This PCR with these particular primers have always worked fine for me. No problems until recently. I am using a mix of fecal DNA as well as DNA from tissues as a control. If you look at the top row, I've got 2 negative controls (H2O) at the end. They look no different than my samples. What could be going on?
Because your target PCR product is ~200 bp, my guess is that the uppper "smear" band in the gel is template DNA that is migrating >1,500 bp. If you run the same sample on 0.8% gel you can find out the exact size. To resolve this issue, you need to start everything with fresh reagents (the current reagents and water are likely to be contaminated with DNA; source of this DNA could be a growth in reagents) and get a better estimate of template DNA amount before using in the PCR reaction.
You should check if the template DNA is desirable. When DNA is to large it may appear at top. So need to decrease the concentration of template. The two marker showed the complete migration on control bands, so I think the problem is not gel concentration. My question is: did you use the same primers before? If yes. The product appeared at 200 bp before? Waiting your answer. We have same results before.
Yes, I have used these primers on numerous occasions. I just tried the reaction again with water that came from the Qiagen multiplex PCR kit (as opposed to MilliQ H2O). The PCR is running now. I also tried a 1:10 dilution of template DNA.
I don't normally check DNA concentration, I usually work with fecal DNA which looks wonky on the NanoDrop we have here. I will try quantifying with the tissue samples and just working with those until I resolve this.
If this new PCR doesn't work, the only thing I can think of is varying the concentrations of MgCl2/BSA/dNTP.
Sometimes it happens in PCR reactions, they stop working. I would suggest you to start afresh, using everything fresh as suggested before and also begin standardization again with varying concentrations of MgCl2 and annealing temperature. Also ensure to take the primers from main stock and not the aliquotes.
hi,
It happens. You can start your work freshly from isolation of DNA to PCR (means you can prepare every reagent freshly and aliquet the primers in fresh tubes, use new (fresh) molecular grade water for PCR and micro-tips and PCR tubes should be sterilize and remember bear Nytrile glove for the molecular purposes, because sometimes without gloves any tissue or dust particle may inhibit the PCR reaction and smear will come.
and use the fresh TAE or TBE buffer and run your PCR product in 0.8-1.0% agarose gel.
if your negative controls cannot be distinguished from your positives then you have contamination of post pcr product in one of your reagents ( possibly carried back from the electtrophoresis area on your gloves) as well as a primer dimer problem which is a separate issue. pcr contamination is very serious as every 10 cycles you get 1000 times more amplified product so it takes over the reaction particularly as it is already small so melts easily and is certain to bind your primers). You must change all of your reagents made up with fresh water from another laboratory and pipettes that have never been used for your amplification to eliminate the contamination.Your negative ( no dna template) control must not amplify. The primer dimer issue could be solved with less primer or by using a hot start enzyme. The smearing of your product probably come from great over amplification of your product which is starting to anneal to other product molecules so gets larger
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