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PCR contamination - (Sep/25/2006 )

I am a Ph.D scholar working on the development of a Multiplex PCR for infectious diseases.
My PCR product size is around 100bp. We have set up a regular PCR with 41 cycles and hybridised the product with the corresponding target DNA. It is observed that there are spots coming even in the product hybridised witht the reagent control (negative control). We have even done a real-time PCR using the same primers and we have observed a product at around 33 cycles (we did a 41 cycles reaction) in the reagent control. We have aditionally treated one tube of the same reagent control with UDG-Glycosylase but still we get a product appearing at 34 cycles (we have shifted to using dUTPs before using UDG-Glycosyalse). We have even tried UV treatment to eliminate the amplicon(s) or genomic DNA, but, with little luck. All my products are of the same size (around 100bps), so, are we getting non-specificity because of the size of the products? We have hybridised both the sample and reagent control products on nylon membranes immobilized with the corresponding target DNA, and everytime we get a good spot in both the products (we use a DAB detection system).
Reducing the number of PCR cycles also has not helped as some of the products failed to show up in the hybridisation due to lack of sensitivity!
We have de-contaminated the laboratory by fumigation followed by formaldehyde treatment and UV exposure, but, nothing seems to work!
At one point, we also have this doubt whether we are actually dealing with amplicons! ( we have not handled these organisms any any point in our laboratory!).
We have in addition, set up the PCR reaction at a different lab by using different labware but, still we encounter amplicons!!!

It would be benificial to me if you could give some valuable suggestions!

-balaji chettiar-

the cause could be sometimes insignificant...the regents used in preparing PCR reactions can affect the end results.....water for example??



Thanks for the suggestions!
Right now my fingers are crossed! I'm going to use a new Mol. Bio. grade water and even subject the PCR product for sequencing!!!

Will get back with the results, sad.gif


-balaji chettiar-