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. Does somebody have some advice what else can I try before going into some special kits enabling PCR amplification of difficult templates? I found several such a helper kits but want to be sure I did all possible before buying some of them.
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!
Edited by Paja, 15 September 2004 - 01:14 PM.