Bsp: condition optimizing - (Aug/04/2005 )

I am working with 10 genes and the msp pcrs for these genes are working just fine, however, the bsp are not working at all. I have already tried multiple different conditions such as:

Nested PCR
different taqs (hotstart: jump start, hotstart taq from Qiagen)

nothing works at all. Browsing the different topics already existing in the forum didn't help either, as I have already tried the suggested taq polymerases.

My sequences are between 300 and 500 bp. So I am actually wondering that HotStart Taqs don`t work as reported in this forum.

I perform my MSP-PCR with a normal Taq from Qiagen using Q-Solution and they work very well, but BSP is driving me crazy!

Please send your advice/suggestions.

My primers for BSP are created by Methprimer.

I am using Hotstar Taq from Qiagen to amplify BSP, and it works very well. Do not use Q solution, since it is for CG rich fragment, not suitable for BSP(am I right, Nick?). Have you ever tried WITHOUT Q solution?
Or you can try other primer sets?Maybe it is because of the primer...

I am not an expert in DNA methylation, just tell my poor experience got recently. I have also tried a lot of Polymerase, and finally decide to use Hotstar...

Good luck to you!

The problem lies in your primer design, you are using methprimer,

can you post the methprimer output for all to have a look.

I routinely perform BSP with Promega Master Mix, works fine, you don't really require a hot start Taq at all.

Haiyan is correct, it is not necessary to use Q-solution for BSP, and MSP for that matter as bisulfite treatment reduces the GC-content of the template by converting all non-CpG C's to T.

Nick

Where your samples from? If paraffin-enbedded tissue, 300-500 bp my be too longer, a primer set for 100-150 bp will be better. good luck...