PCR contamination false positives - (Mar/24/2006 )

Hi

I have a problem. I have been experiencing false positives in my PCR reactions. The following steps have been made to clean up the PCR reaction:

1. All of the pipettes and PCR tubes were autoclauved (we are using new filter pipettes tips).
2. Our PCR water (DNase, nuclease, RNase free) has been aliquotted into acid washed, autoclaved 5ml glass bottles which is then re-autoclaved before use.
3. All new reagents have been used.
4. All new primers have been used.
5. New PCR tubes were tested.
6. New lab coats and gloves were tested.
7. The reactions (and primer dilutions) have all been set up in a lamina flow.
8. Different thermocyclers have been used.
9. Different pipettes and tips have been tested.
10. Reactions have been set up in several different PCR free rooms.

And we are still obtaining positive negatives.

What makes this more interesting is when this PCR is set up and DNA from one of my microbes is put into a positive reaction there appears to be inhibition of the reaction (ie. the negative control is positive and the positive control is negative). This is not happening for any of my other microbes and is not caused by too much DNA in the reaction.

We are now going to sequence the negative to try and find out exactly what is causing the problem.

Does anybody have any suggestions?

you are concerning about materials, but seems not by solutions (buffer, enzyme, primer...)

Seems you have taken much care for preventing contamination.

Since you are working with bugs, i wonder what DNA you are amplifiing... (eg very conserved DNA or from a species closely related to E.coli? Since Taq DNA polymerase is cloned and isolated from E.coli I have heard that there is often some DNA contamination in the preparations. Is it possible that you are amplifing this?

could you post a picture, please?

what size is your contaminating band?

I am working with primers that are specific for only a handful of organisms (E.coli and pseudomonas will not be amplified by these primers). The forwards primer is a universal rDNA 16S primer and the reverse primer has been designed specifically for one of my organisms. I am working with a number of microbes and am only haveing this problem for one.

My products are ~350bp in lenght which is the expected size for these primers. (I will post a picture for you on monday)