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qPCR contamination problem


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#1 joy123

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Posted 29 August 2012 - 02:14 PM

Hi, I have contamination in NCT for long time. I carry out everything under fume hood now, and changed a lot of things. It is better. But in my recent qPCR with 96-well plates, I found that for the 2 NTC, 1 get PCR product (same melting point with samples).

I wonder how this may happen? Please give me some suggestions, so that I can improve it.

Besides, I feel it really hard to totally avoid contamination with 96-well plates. I am thinking, as the air flow is from lab room into fume hood, will the air turbulence make it easier to contaminate as the wells are not covered during adding templates? Is there a method to avoid it?

Thanks!

#2 bob1

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Posted 29 August 2012 - 02:42 PM

A fume hood is not helpful for setting up PCR. A laminar flow cabinet (filtered air blowing towards you) may be, so long as you understand the principle that *anything* you place closer to the vents than the plate you are working on is a potential source of contamination.

Contamination is a very difficult thing to get rid of. It is quite likely that your contamination is from PCR product from other reactions that is floating around the lab in aerosolized form. You should throw out all your reagents and clean your pipettes out thorughly, use filter tip if you can, as these stop DNA from entering your pipettes.

If possible it is best to have a set of pipettes that are used only for setting up reactions (NOT for the DNA though - keep a separate one for this) and have a separate set for loading PCRs onto gels, that are used for this purpose only.

#3 phage434

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Posted 29 August 2012 - 05:34 PM

I think contamination from the air is quite rare. Most likely the problem is contamination in the reagents or water. I would be using barrier tips and make sure that my pipettors were clean.

#4 joy123

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Posted 30 August 2012 - 01:46 PM

Thanks! I use tips with filter. And I wipe pipettors with DNAzap from ABI before adding samples. It really bothers me in the recent months.

I think contamination from the air is quite rare. Most likely the problem is contamination in the reagents or water. I would be using barrier tips and make sure that my pipettors were clean.



#5 bob1

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Posted 30 August 2012 - 02:21 PM

You probably need to clean the inside of the pipettes too.

#6 Trof

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Posted 31 August 2012 - 06:21 AM

Biggest problem with plates can also be carryover.

If you use one of the common master mixes for qPCR, they contain dUTP. If you run the same assays over and over again and open the finished reactions, you may have contaminated the enviroment by previous product. UDG treatment before each reaction can prevent this.
However more often is contamination by the template itself where the UDG won't help. But you can probably try it to identify the source of contamination.

I never really get the point in wiping the outsides of a pipett, but that won't hurt I guess. The insides are however most important, some pipettes have autoclavable shafts/tip holders.

If you now get consistent contamination you may have already one of the solutions you use contaminated. So you have to clean and change everything possible (pipettes, box,...) and change all reagents that can be changed to be sure you start anew clean. If you not using PCR water from a kit, make sure it couldn't get contaminated before you use it (or by "sterilisation" itself).

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