optimizing PCR for low Tm primers - (Jun/25/2007 )
I need help with putting PCR theory in to action. I have a 6 plasmids each with a different shRNA and I have had problem sequencing through the sense strand of the hairpin because they have very high GC content. This fact is its own problem. One idea I had to bypass the secondary structure created by the hairpin was to PCR amplify a segment of the shRNA by having primers created based on the conserved loop of the hairpin. Thus I have a forward and reverse primer from this sequence (along with a corresponding forward and reverse primer in the flanking regions) and I am aiming to have two separate reactions to create a PCR product from each of them (amplifying the sense and antisense portions of the hairpin stem), sequence these products and combine the data to create a beautiful, hassle-free, complete sequence confirming the base-pairs of my designed shRNA. Unfortunately, the Tm of the hairpin loop primers is 50 oC and I have been trying touchdown PCR, only to pick up primer dimers.
So here are my query's:
1) Where should I start my annealing temp at for the TD PCR? 5 degrees above Tm to 2 below --> does this cause the primer dimers?
2) Should I get the corresponding F and R primers, for the R and F hairpin loop primers (respectively), to match the hairpin loop primer Tm at 50 (because they are previously designed primers with normal Tm's around 55)?
3) Should I use any DNA relaxing agents, like DMSO?
4) Should I play with the Mg++ to favour specificity?
5) What else can favour specificity in the context of low Tm primers?
6) Anything else I am not considering?
Any... ANY help would be greatly appreciated because I am stumped with this problem.
you must first try many tm from 50 to 55 (+1°C).
and you can use hight tm (to dicrease hairpin) but in this case you must increase Mg++ concentration more than 1.5mM
because Tm is f(Mg++)