non-specific bands in NTC

Dunowska, Magda via methods%40net.bio.net (by M.Dunowska from massey.ac.nz)
Sun Sep 19 15:45:23 EST 2010


Thanks! I will try this and see what happens - Magda


-----Original Message-----
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Cathal Garvey
Sent: Monday, 20 September 2010 6:39 a.m.
To: WS
Cc: methods from magpie.bio.indiana.edu
Subject: Re: non-specific bands in NTC

REmember to add primers after DNAse.. seems obvious but I make stupid
mistakes daily.

---
Twitter: @onetruecathal
Sent from my beloved Android phone.

On 19 Sep 2010 18:17, "WS" <novalidaddress from nurfuerspam.de> wrote:

Dear Magda,

might be that your polymerase contains traces of DNA from the host it
was produced in. To confirm, add some DNAse to an aliquot of your
mastermix, incubate say 1hr at 37 degC (or overnight if you like) and
then kill the DNAse by heating to 95degC for say 10 minutes (longe is
not so critical, but polymerase of course will decrease in activity).
Then redo your assay. Include a control that demonstrates that DNAse
is really gone; a control plasmid in a reasonable concentration should
be amplified.

Alternatively/additinally, you might try to switch polymerase batch/
brand/type, too.

Hope that helps,

Wo

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