non-specific bands in NTC
(by M.Dunowska from massey.ac.nz)
Mon Sep 20 20:24:12 EST 2010
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!
From: methods-bounces from oat.bio.indiana.edu [mailto:methods-bounces from oat.bio.indiana.edu] On Behalf Of Duncan Clark
Sent: Monday, 20 September 2010 9:12 p.m.
To: methods from magpie.bio.indiana.edu
Subject: Re: non-specific bands in NTC
Historians believe that in newspost
<mailman.185.1284934038.1545.methods from net.bio.net> on Mon, 20 Sep 2010,
"Dunowska, Magda" <M.Dunowska from massey.ac.nz> penned the following
>Thanks! I will try this and see what happens - Magda
And if your pancreatic DNAse is not pure enough it will contain a
protease that nicely cuts up Taq polymerase :-(
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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