problems with MS-HRM - late detection of PCR signal - high resolution melting, methylation (Apr/14/2011 )

Hello,

I would appreciate if you can share your experience.

My MS-HRM PCR signal comes out very late, between 35 and 40th cycle. Otherwise looks fine, but it still doesn't feel right.

I have used 10-35ng bisulfite converted DNA, primers diluted to final concentration 0,3-0,75microM, all combinations. My products are about 150bp long.

I really don't have any idea, what to try further. Same thing happens with purchased bisulfite converted methylated DNA (Qiagen).

Did anyone have similar problems?

Regards,

lmg

lmg on Thu Apr 14 10:46:46 2011 said:

Ok, I have finally found answer to my problem. Ghm, this late amplification is actually normal when using bisulfite converted DNA in MS-HRM. I have realised this when read through a bunch of articles, where they recommend 50-60 PCR cycles... Sometimes the answer is just in front of our nose...

;-)

Have a nice weekend,

lmg

lmg on Thu Apr 14 10:46:46 2011 said:

Hi,

First of all your primer concentration must be 10 micromolar...

master mix=10
primer F:2 or 3
primer R:2 or 3
MgCl= if you put primer 2microL put mgcl=2, if you put primers 3 microL put the MgCl=2,5
H2O=1
DNA=10microM for 10ng

Try it