Hi,
Recently, I have done PCR with 16S primer for identifying my bacteria. I have run my control negative with my primers and without DNA. Unfortunately, I saw a band the same size as my pcr product. Can anyone hellp me? How can I solve this problem? In the control negative without any templte, sould I dont see a band?
This is post pcr contamination.
We have to remember that 10cycles of pcr is1000 time more product and 20 cycles is 1000000 times more product. So even the tiniest amount of pcr product on dust,on the workbench or in a pipette will create huge amplification. You often get contamination by opening tubes or plate seals in your work area and tiny droplets are spread all over your area and the dust can get into the pcr reagents or tubes. Also if you run gels some product runs into the buffer then you lift the gel out of the tank and you get pcr product on your gloves. If you now go back to your work area then touch things then they are contaminated. Also if you use your pcr setup pipettes to load post pcr samples into the gel then some liquid gets past the filter into the pipette and that can then contaminate the reagents when you use the same pipette to measure out reagents.
I would put away or throw all reagents and borrow another researchers taq and all pcr reagents. You will have to order new primers but use new dilutions of the primers made up with another researchers TE or water and using their pipettes and tips not yours. Set up a pcr with 3 no template controls and one positive control or sample. Prepare the pcr using another researchers pipettes,tubes and working area.Use nothing of your own except primers. Also use their pcr machine. While you are running this pcr thoroughly clean your working area and strip down and clean your pipettes. Get new plasticware and clean your pcr machine.. The only sure way to get rid of contamination is to design one primer outside of your primer set but meanwhile lets try to get a clean NTC and a working PCR but it may take time.
Hello, if you are using universal bacterial primer you need to be careful with contamination as from as using surplices as weel from lab environment.
Felipe Alberto Lei
Paul Rutland
thanks by your answer, I have done PCR with one reverse and forward primer separately and I didn't see any band, but in reaction wich have both primer, I see a sharp band. If primer has contamination, should we see a band in reaction with one primer or not?
this is my picture about my PCR. the band wich next to ladder is the sample with template and the another is control negative without DNA
Hi Bahare, in your last coment you said " I have done PCR with one reverse and forward primer separately", do you mean only put one primer in the master mix ? If you want to know if there is contamination of your primers, yo need to work with a new aliquote or new batch of both primers.
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