Homemade RealTime Syb-GreenI-Mix - (Jan/27/2006 )

Hi,
I try to establish an selfmade mix for qpcr with the biorad-icycler on the base of
sybr-green.
Does anybdy have experience with that.......is it possible to get good results from that.

ThanX an Greetz

K_R

wow, if anyone has a recipe I'd be all over that...it's spensive stuff, it is

I try to optimize it depeding on this publication

http://www.cif.ucr.edu/Assets/QPCR/QPCRmix.pdf

it seem to work (the curves are nice), but Ct-values are a little bit unstable....searching
for input,how to optimize it...

no experiences at all...??? :-(

For some reason I suspect that this is not worth the effort...especially if you're interested in accurate publications

-Matt

that's the main reason I haven't tried to cook anything up...it would be difficult to trust my results

OK...I think its a good point to proove your results with an comercial mix....
but I don't want to convince anybody, I just think it is an interesting thing
as a little side-project... and the first result
are looking ...äh....moderate, but improveable
..so let me ask it in a more general way...
what are the most common ways to stabilize a pcr, to prevent taq from loosing activity
during 40 cycles?

ThanX

Reduce denature temperature & shorten denature time will help to keep the activity of Taq
By the way, I remember the dilute PCR mix still work for real time PCR in case of save money, however it is not recommaned for collect data using in publish paper.

Loss of taq activity is seldom a problem if you denature for 15 sec at 94 degrees celsius.

If you understand qPCR, you really only need it for <30 anyway. You'll obtain your ct value before then.

-Matt

OK...I get my Cta around 20 that is good and compareable with the commecial mix...the only problem is the variation between two
identical samples....and they are identical because I made an all-in-one Mastermix (incl. primers and cDNA) and than distributed in 2 wells....with the commecial mix the variation is 0.1-0.2 with the homemade its 0.3-0.6 approx. I thought, maybe it is because of degradation of the taq, because high ct means more variation.
What is the most promising factor for stabilizing the reaction?

K_R

PS: Maybe somebody has experience with homemade Sybr-Green I Mastermix???