How to remove inhibitory substances in PCR? - (Jul/12/2014 )

Hello,

I am trying to take a environmental wax sample (containing a mixture of fatty acids and wax esters) containing bacteria as a template for PCR after emulsifying the sample with Tween-80 in water. The final concentration of Tween-80 in the PCR tube was 0.5% and the concentration of the wax in the tube was 30%. I never got a band after running an agarose gel.

Then I spiked the the wax sample with different numbers of E. coli: 70 to 7.000.000 cells per PCR tube. Again, I obtained no amplicon. When I am doing PCR without the wax, but only E. coli I got nice bands. Hence, there must be some substance(s) in the sample inhibiting my PCR.

I can speculate that lipids of the sample inhibit the PCR.

On the other hand, the emulsion will form kind of miniature reacting vessels in the PCR tubes: small water droplets containing templates and PCR mix. This would shield the amplification reaction against lipids on the outside of the droplets. The fact that PCR is still inhibited leads me to the idea that there is something hydrophilic in my sample which renders PCRs impossible.

Any ideas what one could try to identify and solve the problem?

Two suggestions:

1) you can remove most of the wax and lipid by extracting with chloroform

2) you are probably using far too much of your template sample. You need vanishingly small amounts of template (1 molecule, in principle). Diluting template very oftren solves inhibitor problems.

I'd guess that it's more a problem that the chemicals solved in water never reach the bacteria in a hydrophobic "containment" and the DNA isn't extracted then, so more a separation problem, that can be solved with phage's suggestion.

The idea that wax isn't an inhibitor comes from my experience with DNA extraction from waxy insects such as white flies (they're covered completely with a white scaly wax layer). I use a quite simple salting-out protocol and surely some wax is still in the sample...anyway it's much less compared to your samples and therefore isn't a problem (another difference might be the wax composition).