Thanks for sharing your outcomes Magda, and it's great to hear it worked! --- Twitter: @onetruecathal Sent from my beloved Android phone.
On 21 Sep 2010 04:02, "Dunowska, Magda" <m.dunow...@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 -----Original Message----- From: methods-boun...@oat.bio.indiana.edu [mailto:methods-boun...@oat.b... Historians believe that in newspost <mailman.185.1284934038.1545.meth...@net.bio.net> on Mon, 20 Se... _______________________________________________ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
