No replies to this topic

[mybioweb2]

Hi,
I'll be grateful if somebody could help me. I've a strange thing in my macroarray cDNA probe synthesis.
for a macrarray experiment, I synthesize my P-33 labeled cDNA probe in 20µl using 25µg total RNAs and 2µl of PoweScrit (Clontech) as RT enzyme.

When I proceed to measure CPM before and after probe purification using ProbeQuant G-50 column, to determine the % incorporation, I find that the CPM (Count Per Minute) after purification is much higher thant CPM before purification?!!!!!!! why i've this ? does anyone has a something like this ? what would be wrong?

in spit of I use fresh RT enzyme and all other reagents are ok !!!

is PowerScript RT is less efficient than SuperScript II in radiolabeled cDNA synthesis? or, is the enzyme quantity (2µl < 100 U) not enough to efficiently synthesize cDNA from 25µg total RNA?

any hits concerning this problem, or plant macroarray protocols would be helpful.
thanx in advance.