Milliq water isn't necessarily free of contaminating DNA molecules (especially from bacteria) even after autoclaving. Without purchasing special water that is certified DNA/RNA-free, how would someone go about making Milliq water free of any contaminating DNA molecules?
Would we add DNase, RNase, incubate and then remove them with DEPC and Autoclaving?
I'm specifically interested in amplifying bacterial DNA and want to make sure that my reagents are free of contaminants.
To make DNase/RNase free water using DEPC: In a 100 ml pyrex bottle, add 80µl of DEPC (D5758 Sigma) to 80 ml of distilled/osmosed water, incubate at 37°C for 18 hours, autoclave at 120°C/15 min, aliquote in 1.5/2 ml microtubes, and stock at -20°C.
Does your water need to be all DNA free?. Your test is for specific dna so any other dna is not really important. You could just take a few samples of good quality water and amplify with your specific primers. .Any water sample not amplifying would be good enough for use and could be aliqoutted for future use.
really the initial expense of buying some good quality water along with the other reagents will more than compensate for any time wasted sorting out contamination problems later
Paul Rutland Thanks for your answer. The water needs to be free of contaminating bacterial DNA. I've seen a study that suggests autoclaving at 121C for 120 minutes can degrade DNA sufficiently such that it is not detected via PCR (Article Decomposition of waste DNA with extended autoclaving under u...
).It is nearly impossible to remove bacterial DNA from samples and it is fairly prevalent in microbiome kits and the like.
Extracting a blank is a good control but what are good steps for removing even traces of DNA from my water?
If their method works then adding hydrogen peroxide or even dilute acid followed by extended autoclaving and relying on the pcr buffer to determine the pH but if your pcr is 30 cycles then testing some water samples to find one that has no amplification as a negative control at 40 cycles would seem to be sufficient. I suspect that dna from some extremophiles might still survive at 121c