We're doing RLBs (Reverse Line Blots) which are a cross between a Western and a 2D blot, but using DNA oligos (probes) and bacterial PCR products (run 90 degrees to probes). We are doing a multiplex PCR using a genus-specific 23S-5S spacer primer set and a universal eubacterial primer set. We are trying to determine which bacterial species is present in a sample. We sometimes see one of 2 probes (never any others) lighting up on the blot in the no DNA lane, but NOT in the other lanes. We've tried fresh water, wiping with 70% EtOH and a Nucleoclean solution and UV irradiation. We've run out of ideas and could use some help.
Hi Lisa! I guess you UV all your PCR sample mix except the DNA and the polymerase, then the only source of contamination I can think of is the enzyme preparation. Remember that they have a bacterial origin and there is always some DNA that might be present. You could try using DNase I on your sample mix but don’t put primers. Heat-inactivate and proceed with your PCR.
It would be very interesting if you sequence those bands in the negative control.
Hi! I meant you incubate your Taq polymerase with DNase I and then heat-inactivate it.
Use Milli-Q / RNA-free water. Also spray the table with RNAse Zap. You can also try using fresh reagents for the PCR.
When dealing with contamination, it is best to start with all new ingredients of the PCR mix and discard all the old reagents. This includes primers, probes, dNTPs etc. If you have already done this and you still have problems, Abdelhalim may have the solution.
A likely cause of contamination when using universal eubacterial primers is amplification of E. coli DNA that is contaminating your PCR mix. The Taq in your PCR mix is generally produced from recombinant E. coli cells and some nucleic acid can be co-purified when producing the enzyme. One solution, apart from that presented above, is to purchase a PCR mix where the Taq is purified from T. aquaticus and not E. coli. Takara used to supply such an enzyme but I am not sure if they still do.
I hope this helps.
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