Home-made Sybr green mix for real-time PCR - (Feb/10/2006 )
I have prepared home-made mix with 1:25000 (working) from stock and it works good.
I prepared the dilution of 1:1000 (10x) from 10000x stock by 2 steps.
First, I prepared the 1:10, then I use 1ul to prepare another 100x fold dilution, so I got the 1:1000.
However, I encountered problems when the 1:1000 aliquot was used up and I prepared it again from 1:10. The fluorescence signal for the new batch dropped 10 fold , from sth around 1800U down to 180U.
Anyone would give me some advice and help me to solve this problem?
Or suggest me on how to stabilize the Sybr Green aliquots
(what I suspect is the diluent for SG, as I read sth using TE, but I forgot to do so when I diluted it)
what are your storage conditions?
I also read about TE Buffer as the best
I think the buffer is important for stabilizing
the fluorescens is also not absoluly linear with the
ammount of active Syb. Green
I once did 2 runs with to compare the effect
of doubled SG-ammount
x1: max. at 14000 Units
x2: max. at 70 000 Units!!!
so maybe you have lost 50% activity (which maybe
normal in longtime storage) but have an decrease of
1/10 in the signal....
maybe you just double the ammount of SG-Workingsolution
and look what happen to the signal....