ishmistry

 MSP template and primer seqs.docx   124.54K   84 downloads
Hi,
I've been trying to do MSP for months now and just can't get any products from my bisulphite treated genomic DNA.
I've been trying to amplify a region of the HIF1 promoter to repeat some work done in this paper:
http://www.nature.co...nc2010481a.html
But there is no protocol given in the paper and the reverse primer they say they use, i can't find anywhere in the target sequence.

I designed my own M and U primers using methprimer but cannot get products from my bisulphite treated genomic human DNA template with either primers. Tried another set of primers that i designed by eye but no luck either. (target DNA sequence and primer sequences attached).
I've tried Gotaq polymerase, Q5 hot start polymerase and KOD hot start polymerase. I've tried a range of annealing temperatures (40-65C), primer concentrations: 0.2-0.4 uM and input template amounts: 100-300 ng.

To add complication, i was getting amplification of the region 100bp upstream from this using primers designed for bisulphote sequencing, sowing my template isn't the problem.

I'm at a total loss, can anyone help?

Thanks

Ish