Negative control of one of the primer I used to do RAPD analysis seems contaminated. Can someone tell me about where the things get wrong?
there are many. If the contamination is weak then the negative control may be contaminated with bugs. Autoclaving the water kills bugs but does not destroy the dna so the contamination remains. If the contamination is quite strongly amplified the reason is probably contamination from previous amplifications. Every 10 cycles of pcr gives about 1000 times as much amplimer as was in the original sample so even a tiny amount of pcr product amplifies very well. It is usually transmitted from the running buffer of a previous gel and from handling the gels to visualise them. The buffer with some amplimer in it dries on the gloves and blows around the lab as dust or just you handling pipettes or plasticware or reagents will contaminate them. Ideally gels and post pcr handling should happen in a lab separate from pct reaction lab to stop transmission of post pcr product into the reagents and working areas but this is not always possible.
Use new aliquots of all of your reagents ( borrow from another researcher if necessary ) and water from another laboratory for reagent make up and for the negative control. wash your working area thoroughly and clean all your pipettes and be very careful about returning from post pcr areas to leave your gloves behind in the gel running area. Sometimes contamination can be minimised by UV irradiation of reagents and equipment in tissue culture cabinets or on transilluminators but I usually find that changing all of the reagents works even if it means getting the primer re-synthesised .
Dear Paul,
Thank you very much for your valuable suggestions. Definitely I'll use them in my next PCR.
Hi,
Prof Paul has explained in detail the possibilities of contamination in PCR and how to get rid of them...See that you use filter tips particularly when adding template and primers...If one is not careful the contamination builds up over the time in reagent tubes due to aerosol contamination..
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