PCR BLOck contamination? - better anti-contaminant PCR - tube? (Sep/18/2006 )
Recently I have experienced PCR contamination. Water control got a same size band as positive, but the interstity was stronger that positive control's, and sometimes was weaker than postive control's. Then I set up the reactions at other's lab using my own reagents primer and plasmid DNA only; Run them in my PCR machine and other's machine. The water control got the same size band as positive control using my machine, but the water control in other machine gave a very faint band at the begining of running -gel, (10m later it dissappeared). This experimet indicated
my primer contaminated with plasmid DNA or /and amplicon very lightly, the major contaminants was PCR block! The thin wall tube is made of PP which is not permeable to vapor), but it easily change shape even with finger pressure., this might allow the contaminants get through the tube .
Has anybody had a same problem with PCR block or tube, any suggestion is very welcome!
A very strange occurance. I have never heard of such a form of contamination before.
I do however have to point out that there are variations between models of thermal cyclers, which would result in difference responces for the same PCR mix and cycling conditions. This variation is precision of thermal control, speed of temperature cycling, themperature stability etc...
Rather then contamination getting in between the capped lids of the PCR tube, could your contamination be better amplified on your home lab machine and less well in the machine from the other lab.
Is there anyway to bin your working primer stocks and use a new batch of working primer solution? If you can kill the contamination there, maybe the problem will go away.
Nevertheless, if the PCR block is suspect, a simple treatment of Trigen, followed by a bath (if the block is detectable) in warm acid or bleach. Followed by lot of water.
If none detachable, well trigen, water wipe down, DNAse solution wipe, leave for 1hr or more, wipe down again with water, and again and again.
Run PCR reaction. And see if that helps.
What I saw on the gel was the 2 positive control on the different machine had similar intensity.
My block is not detechable, but what I heard is bleach and DNAase solution might ruin the machine.
I myself also do not believe the block contamination very much.
What I have to do is to reply the primers(amplify different region) and other reagents, and see what will happen.
Thanks.
perneseblue:
My observation might be explained in your way, but I have a feeling some strange problem often happenes against people's intuition. I hope I am completely wrong, otherwise PCR contamination will kill me soon or later!
What I should have done is to set up the reactions in my lab using all of my reagent and run them in the different PCR machine, this result might be more helpful.
My observation might be explained in your way, but I have a feeling some strange problem often happenes against people's intuition. I hope I am completely wrong, otherwise PCR contamination will kill me soon or later!
What I should have done is to set up the reactions in my lab using all of my reagent and run them in the different PCR machine, this result might be more helpful.
And don't forget to do the reverse expt: set up in the other lab. Actually, set up two sets of tubes, seal them and run one set in each lab.
You should also consider cleaning your pipettes (if your lab is like most uni labs, that won't have been done for a loooong time). When you did the expt in the other lab, did you use their pipettes, or did you take yours and use them?
Swanny,
That time only plasmid DNA and primers is my stuff. I used other's pipettor, tip, dntp, taq.water, tube.
Swanny:
use foil to seal them plz?
use foil to seal them plz?
Foil? Speculator, are the tubes being sealed by foil? Could the foil be contaminated. If not, could tube caps be used instead? Or am I just mistaken here.
perneseblue
No, they were not sealed by foil. I am going to seal them next time.
In my Roche hand book there is a protocol to prevent crossover contamination with previously used samples. I have never tried and it sounds quite expensive:
They suggest to add to all reactions dUTP and uracil-DNA N-glycosylase (UNG). dUTP is incorporated during PCR in the amplicons. The UNG kills contaminats from previous PCRs if the PCR mix is pretreated with it and is itself later denaturated by heat. So it cannot serve as template. The newly template you use contains thymidine and is not affected. But it does not help against genomic DNA contaminants.
It is also available as kit.