thanks a lot methylnick....i jus finished my MSP., got no bands in any lane...i explain you how i did...
i selected a normal cell line tat express basal level of my gene of interest as a comparison control for my gene tat express more in cancer cell lines. so first inorder to optimize MS-PCR i used three points, one is the DNA of normal cell line bisulphite converted (200 ng- qiagen epitect Bis conv. kit- eluted 20 micro litres final, from which i used 6 micro litres for PCR),second is the non converted same cell line (used 25ng Dna for PCR), third is unmethylated control supplied by qiagen (10 ng -suggested by qiagen).
The only change i did was instead of 50 Micro litres, i used 25 final volume for PCR, i dnt know whether this could be the reason, bcoz on seeing the gel it sounds like PCR failure, as there is no band in the Unmeth control itself.
PCR Reaction: (qiagen MSP kit)
Template : 6 micro litres (from 200 ng bis converted and eluted 20 micro final, i dnt know is it possible to quantify the bis convted Dna, so )
Prim U/M F : 0.4 micro molar final conc.
Prim U/M R : 0.4
Msp Master Mix(2x): 12.5 micro litres
Water : to the final 25
x 35 cycles
Amplicon size 110 bp.
Kindly suggest me to optimize the protocol for MSP. also enlighten me which all points to use; i mean samples i can use for PCR optimizaton alone, may be is the meth or unmeth control alone is enough for optimization or do i hav to run my bis conv Dna also along?.
let me kno if i need to provide any other details..
thanks a lot for any of ur suggestions...
is it right to amplify the promoter region of my gene of interest and then continue with the bisulphite conversion and MSP or hav to do with the whole genome only???
Edited by GNANA, 17 September 2010 - 11:01 AM.