non-specific bands in NTC

Cathal Garvey via methods%40net.bio.net (by cathalgarvey from gmail.com)
Tue Sep 21 02:58:11 EST 2010


Thanks for sharing your outcomes Magda, and it's great to hear it worked!

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On 21 Sep 2010 04:02, "Dunowska, Magda" <M.Dunowska from massey.ac.nz> wrote:

Hi everybody, thank you so much for all your thoughts and suggestions - very
much appreciated! Just to update everybody: my DNase must have been clean
enough, as it didn't chop up the Taq polymerase, and the treatment got rid
of the non-specific bands! I use Roche 2x master mix, and I treated it with
DNase before addition of primers and water, so obviously whatever was
causing the problem was in the ready-to-use mix, rather than anywhere else.
I like the idea of left-over plasmid from Taq expression, but I guess it
could also have been a contamination that happened at my lab - I can't
exclude this without further investigations (e.g. cloning and sequencing). I
went back through my records and realised that the same thing was happening
on-and-off with other primers too (same 2xmaster mix), so it is not
something specific to one primer pair. I am nearly at the end of the current
batch of this mix, so I'll let it be and see whether or not I have the same
issue with the new one !
 I've ordered. In a meantime I've increased the annealing temperature from
60 to 65 deg C (I use 15 sec annealing time, so I think it's short enough as
it is), decreased primer concentrations from 0.5 to 0.2 uM and decreased the
cycle number from 40 to 35 cycles, and I now have clean NTC lanes (and still
plenty of the specific product in positive lanes), which makes me very
happy!

Thanks again for everybody's help!
Magda



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