User error or pipette contamination - (Oct/30/2013 )
My work colleague ran an experiment and his water control, all four of them came back positive. Now the intriguing part is that the bands had an identical intensity to his positive wells and the bands were also the same size.
He has decided to lay the blame on contaminated pipettes rather than making a mistake. The pipettes are general purpose and are used for all sorts of reagents and DNAs, filtered tips are also used.
So what is the likely hood of not one but both primers binding onto foreign DNA and also amplifying a fragment of DNA that is an identical size to his target DNA? Not only that but if the pipette was contaminated, the DNA happened to be identical to the DNA being used in his experiment.
With all these coincidences what, out of contamination and user error, which do you think is more likely?
perhaps one of the other chemicals (water, buffer, ...) is contaminated with the sample DNA not the pipette?
This situation definitely sounds like an instance of contamination, whether its the water, buffer, or pipets. qPCR is notoriously sensitive to contamination, and some labs even carry out master mixing in a sterile work space/hood. Often the PCR machine is also inside the hood.
PhalanxBio on Wed Oct 30 20:36:52 2013 said:
Often the PCR machine is also inside the hood.
What purpose does it have?
I suppose you only put closed plates/wells in the machine, so there is no way how any contamination could get inside around the machine even if it was contaminated. And the machine actually is the place where the 'sensitive' become 'contaminant' within hours, but the same as before, you should be opening the wells anywhere near it too.
Unless of course you contaminate gloves while handling the machine but then I don't really get how it would get so awfully contaminated in the first place.
We pipett PCR in the hood, then close, put in machine, put out and open and analyse in a different room. I would consider having cycler in the hood as a very bothering.