MSP Primers not working - (Apr/20/2012 )
This is my first post here. I'm looking to see whether methylation is occuring in the promoter region of the bax gene, so i've found the promoter region;
Then have used the MethPrimer Program to design Primers which are these;
MS: GGG AGA GTT TAA ATT TTG TTC G
MAs: ATA CCA ACA ATA ACG CCG TC
US: GGG AGA GTT TAA ATT TTG TTT G
UAs: AAA TAC CAA CAA TAA CAC CAT C
I've tried different annealing temperatures, and have tried them on bisulphite treated DNA, and that which hadn't been treated. Still no bands visualised when i've run a gel. Any ideas where i'm going wrong or what I could try next?
i haven't checked if your primers are OK (technically), but would recommend if you tried Q solution from Qiagen.
I had problems with one product, and this Q solution helped. I use it with HotStart polymerase.
I also deeply recommend using the HotStart Taq poly (Qiagen) and Q substance. I used 2x Gold Master Mix, Applied Biosystems and have never got the bands. Situation has changed with Qiagen HOtStart Taq and Q.
Thank you! I'm also going to try a gradient PCR and see if that works. Thanks for your help
If you want, inform me on your results
Armwaly, it was just Ampli Taq Gold Master Mix (http://tools.invitrogen.com/content/sfs/manuals/cms_041165.pdf), it was not 360.. I hope your MSP experiments would be ok!