I am trying to amplify (and eventually sequence) promoter region of my gene. The problem is high content of GC (73%). I tried almost everything possible and now I am without any new idea.

The whole amplicon is supposed to be 507 bp long. My primers were designed using Primer Express Software, forward primer has length 19 nucleotides, %GC 63, Tm 60°C, reverse primer is 20 nucleotides long, %GC 60, Tm 60°C.
The whole amplicon has Tm 89°C, Ta 65°C, and as I already mentioned % GC 73.
I am using AmpliTaq Gold polymerase (1u into 20 ul reaction), 3.5 mM Mg2+, 1000 nM final primer concentration,
First I ran my “regular” PCR cycling conditions: 96°C for 13’, than 35 x 96°C for 20’’, Ta for 15’’, 72°C for 30’’, final extension 72°C for 1’. Ta: 58°C, 60°C and 62°C. All what I got was signal at annealing temperature 58°C: 1 weak band around 300 bp, another one around 1000 bp, and two very weak bands 200 bp and 400 bp…

Then I changed cycling conditions the following way: 95°C 13’, than 35 x 96°C for 40’’ to allow complete denaturation, Ta for 40’’, 68°C for 2’ and final extension at 68°C for 10’. Ta: 54°C, 56°C and 58°C. Simultaneously I had serial dilution of primers: original 1000 nM, 600 nM and 200 nM – I have read that high primer concentration may lead to formation duplex (of course) that traps all the polymerase. And because I tried also 3 concentrations of DMSO (0%, 5% and 10%) that inhibits Taq polymerase I assumed the trick with lowering primer concentration may help…
And I got the following:
200 nM primers: 0
600 nM primers: terribly weak band around 110 bp when using 5% DMSO, but also in NTC so I assume this is just primer/dimmer/whatever mix
1000 nM primers: weak band around 110 bp everywhere (also in NTC) regardless Ta or % of DMSO used.
So, please, any suggestions?
Thank you so much in advance!
Paja
Edited by Paja, 15 September 2004 - 01:14 PM.