False priming in MSP - (Jan/30/2007 )

Hello everyone,
I’m having some difficult to define primers for MSP to the gene that I’m studying.
How can I prevent false priming or dimmers in my reaction? How that could be a problem in Real Time PCR using SyberGreen? Could I have false positive?
Regards,

-brstudent-

Hi brstudent,

how are you picking your primers? By eye or using a software? AB Methylprimer Express does a fairly good job.
MSP is based on primers differing in just one base ... so false positive and negative findings are certainly an issue. Most accurately, you have to validate your primers either by BSP or by creating standard curves using several mixes of unmethylated and methylated DNA (like 0, 10, 50, 100% methylation).

Good luck!

Krümel

-krümelmonster-

QUOTE (krümelmonster @ Feb 1 2007, 06:42 AM)

Dear Krümel,
Thank you very much for your answer, I will try this way.

Now I have more questions, could you please help me (or anyone else)?
How can you create a mix of unmethylated and methylated DNA? Do you use SssI and S-adenosylmethyonine? Could you please give me any contact to buy S-adenosylmethionine? I'm having some dificult to find this product in Brazil.
Regards

-brstudent-

Hi brstudent,

I am using SssI and SAM and I get it from New England Biolabs. SAM is supplied in the SssI package. New England Biolabs have the following distributor in Brazil:

UNISCIENCE DO BRASIL
Av. candido Portinari 933/937
05114-001 São Paulo- SP
Brazil
Tel: 11 3622 23 20
Fax: 11 3622 23 33
e-mail: info@uniscience.com
Web site www.uniscience.com

Good luck!

Krümel

-krümelmonster-

Hi Krümel,
Thank you very much for helping me.
Regards
brstudent

QUOTE (krümelmonster @ Feb 2 2007, 08:11 AM)

-brstudent-