Feather microbes - PCR troubleshooting (Sep/17/2004 )

Hi,

I'm new to this forum so I hope I'm doing this right! I just posted this same topic in the microbiology section.

I am involved in a post-doctoral project looking at microbial diversity on feathers. We are currently attempting to amplify bacterial DNA directly from the feathers (no culturing) using conserved primers and are having some difficulties. We are essentially getting very little or no product at all. Lately, the PCR products have been smeared. Would anyone have any helpful comments or any suggestions? We are starting to wonder whether there is simply just very little bacteria on the feathers.

Thank you in advance.

feathers

Hi,

do you have some non-degenerate primers for at target you know is there? This might be a good test for your sample preparation procedure.

How degenerate are the primers? If they are very variable, each specific primer is in very low concentration and the many primings can have MANY things going on in your reaction.

It may help using less degereate primers and decreasign specificity by increasing salt or lowering temperature ( maybe only in the first cycles ).

good luck,

regards, Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

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