Hei everyone,
I ll start here a discussion for which I can't find any answer online. We are facing an issue with the water that we use in PCR and qPCR. The problem is that when we use 16S bacterial or universal primers, the background water contamination is very high, with no template controls typically emerging at around 24 cycles on qPCR. As a comparison, using archaeal 16S primers or any other functional gene would typically have the negative control emerge at 38/39 cycles on qPCR. 24 cycles is too low for some of our low biomass samples, so how can we decrease this background contamination from the water?
Here is how we prepare our PCR water:
- Start with milliQ type 1 water that has >18Mohm resistance.
- Filter at 0,2um.
- Autoclave. This is likely useless, as it will kill whatever remains, but not really impact DNA strands.
- Aliquot in sterile 1.5mL tubes.
- "Burn" under UV light for a while.
The main improvements I think could be to use 0,02uM filters, and to increase the UV exposition (strength and time). But does anyone has other suggestions?
As well, does this discussion make any sense? At this point it feels like a rather thorough protocol, and maybe the contamination rather comes from the polymerase kit/buffer or our tubes? What are your thoughts?
Finally, I can say that in the past we have managed to have negative controls on bacterial 16S qPCR emerge at 27 cycles... corresponding to around 10x less contamination. So it is possible...
Thanks for the help!
I think that both filters and ion exchange resins have a similar problem in that high salt concentartions can build up locally on the membrane/resin which is food for bugs so good conductivity but poor purity which is a problem for pcr. You might try heat distilling or just buying a bottle of commercially available pcr grade water and storing it in aliquots
you can simply use nuclease free water.... or try treating your water with DEPC...
Paul Rutland and Keshav Meghwanshi , thanks for your answers. We get PCR-grade water each time we buy the polymerase kit and this water does not perform better than our self-made one.
The point of DEPC is to remove the enzymes, not the DNA strands themselves. Also, Thermofisher does not advise it: https://www.thermofisher.com/order/catalog/product/R0601
Sven Le Moine Bauer . You may want to examine where water could get into your pcr, So dissolving the primers and dNTPs are possible sources of contaminated water entering a pcr reaction as well as the addition of water to make up the volume so these may need replacing. What reason do you have for blaming the water for these false positives? It seems possible to me that you are just seeing a post pcr contamination problem amd that your pipettes , working area or any reagent common to all samples could be to blame. It might be interesting to set up a pcr in another area with different pipettes and plasticwae and another researchers reagents and a newly thawed aliquot of primer and see if the resukts improve
Paul Rutland, I should have been a bit more explicit about our protocol.
There are 4 reagents going into our qPCR mixes:
- DNA template (extracted from something, eluted in water). Absent in our no template (negative) control.
- Primers (ordered dried, resuspended in water)
- Quantitect master mix from QIAGEN (contains buffer, polymerase, sybr green and dntps)
- Water
We have no control over the quantitect master mix. We use it as we receive it. Our water is however involved in the 3 other reagents. So yes, you are right that we may need replacing our primers and water stocks, but therefore I ask: How do I get cleaner water? It isnt a problem that we see just now in the last batch, but since several years.
I think it is difficult to make our preparation protocol cleaner: We prepare our mixes in a sterile cabinet that is cleaned with UV light after each use. The pipette tips are bought sterile and with filters. The tubes are sterile and exposed to UV light regularly (kept under the cabinet). For these reasons I would blame the water rather than something else. But you are right, I could try to make the mixes somewhere else.
Hi Sven Le Moine Bauer ,
I agree that there is nothing you can do about commercial mixes but I do not expect there to be a problem with these.
I would buy /collect some pcr grade water ( from kits/enzymes or commercial company)and use this water to elute a couple of dna samples . Also make up the new primers with this water and use it to make up the volumes in the pcr reactions. Include 2 no template controls in the pcr with the new water. One has old water as NTC and the other has new water as the NTC
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