environmental DNA gels

chrisroesch chrisroesch at okay.net
Wed Feb 9 15:09:20 EST 2000


Hello Danielle,

I'm also working with environmental sample DNA (but in my case with a humic
soil). In my PCR with _nifH_ primers (which are only degenerate in 2 and 1
positions for forward and reverse) I get smear too -- if i get amplificates
at all. But the products I get are always the right size.
Maybe you want to redesign your primers? Consider that in _nifH_ there are
many regions which are very conserved -- perhaps your primers are much too
degenerate?

For _nirS_ there are also quite good primers, _nirK_ may be a more serious
problem (currently we are trying to improve our primers as they give
amplicons from to few positives).

The smear is, I think, nearly inevitable. I rarly have seen PCR photographs
which show one clear, sharp band if the template came from DNA isolated from
soil.

To improve specifity you also might want to try adding 1% formamid to your
PCR mix, or rise [MgCl2]. Another alternative might be adding T4 Gene 32
protein (from Roche) at about 1ug/ul, which increases the Taq's resistance
to impurities of the isolated DNA (e.g. humic acids in soils, other high MW
phenolic compounds or polysaccharides)

(a note: if you want to get fine pictures, stain with EtBr at low conc.;
sybr dyes are far more sensitive and give you strong signals even from minor
co-amplificates)


Hope I could be of help.

Christopher Roesch
University of Cologne
Germany







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