PCR contamination - (Dec/26/2015 )

How to discover the source of contamination and get rid of it?

Hi guys,

I work with ISSR markers. I am struggling a problem for more than 2 months. Consistently the negative control in the PCR reactions are showing contamination. I alread changed the reagents twice, including the water. I always autoclave the tubes and pipette tips. I do the PCR reactions in the flow. Before starting, I place tips, gloves, pipettes, water and the tubes for 15 minutes in UV; nevertheless, I still get bands in the negative controls and in all negative control displays a different pattern of bands. I have already used new reagents, exchanging one at a time, tubes, pipettes, water. I do sterilize the flow with bleach. However, I have not found out the source of contamination. The worst is that in the same reaction of negative control, using the same mix, some reactions appear bands and others do not.

Does anyone have any idea what is the problem? Any solution?

Thanks

I'd strongly recommend that you STOP autoclaving your tips and tubes. And water, if you are doing that also. Autoclaves have junk in them, and almost all tips and tubes are clean.

I agree with phage34. 

Moreover, there may be contamination in the enviornment which could affect your qPCR. If your NTC values are 35 and above then its ok as anything over 35 is not considered in qPCR calculations.

For PCR reactions always use tips with filter and already sterile. Use PCR grade water, all things dedicated to PCR only (pipets, tips, tubes, etc). Instead of bleach that is a corrosive use a cleaner that is for nucleic acid/nuclease contamination. Nucleic acids and nuclease can survive the autoclave thats why Phage doesn't recommend it.  I use all disposable plastic that are already sterile, others use a mix of plastics and glass where the glassware is clean by a serial of baths (acid, base, soap) and for sterilization instead of autoclaving they bake at 400C to eliminate any nucleic acid/nuclease traces. 

Our lab had this frustrating issue, too. New primer stocks, new buffers, new filter tip boxes, even new polymerase tube didn't remove the issue. We found that performing the same PCR experiment on the lab bench of a lab member who works on something completely different solved it, though. The cDNA was the same and the PCR reagents were the same, but somehow the different bench and pipette set made the difference. Using filter tips should prevent junk from the pipette from getting through, but maybe they didn't help, or else the junk was in the metal part that ejects the tips? I always wiped down my pipettes with ethanol and RNAzap before RNA extractions. I thought they were clean.

My advice: Try moving your work area and using someone else's pipettes. See if that helps and let us know what solves your problem.

It's good thing to have at least a so called PCR box if not the hood for doing PCR. I know that people do it on the benches all the time, but that is an enviroment you can never fully troubleshoot. If it works it's good, when it stops working, you are screwed. Maybe a bed venting or whatever, you may never find out.

PCR boxes aren't that expensive, it's just a plexiglass box with integrated UV, you can clean it, keep it shut, keep pipettes there, use UV regulary.. and you won't need a fancy NA degrading stuff, just usual detergent and ethanol. And filtered tips, of course.

(it also helps if pipettes aren't "someones" but rather for a specific task, so they are not changed inbetween them, but I got it long ago, that this is just how it works elsewhere..)

Do you check the loading buffer? I encountered this problem before, there was no problem with other things, just the loading buffer got contaminated.