real time sybergreen concentration-Real-Time PCR
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any body known realtime sybergreen concentration in pcr reaction.
i planed external addition all sybergreen kit (roche) kontent???
the only real advice i can give you is to not add sybr green externally, but to buy it in a kit - like Sigma SybrGReen jumstart ready mix which is what i use (lots of companies have these kits eg. sigma, invitrogen). only because it reduces the error you may have in adding it seperately. and with real time, it is so sensitive that reducing any errors (eg. pipetting error etc) is extremely important. it also makes it easier to compare between experiments.
other than that, maybe look up these kits anyway - they might have what concentrations they use in them. or contact the companies and ask them (although it might be proprietry information).
SYBR Green I is available as a 10,000x stock. The final concentration in most QPCR reactions is 1x.
Hi!
Maybe these two papers can help you (I found them recently):
- "A quantitative fluorescence-based microplate assay for the determination of double-stranded DNA using SYBR Green I and a standard ultraviolet transilluminator gel imaging system.", Vitzthum F, Geiger G, Bisswanger H, Brunner H, Bernhagen J., Anal Biochem. 1999 Dec 1;276(1):59-64
- "Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications.", Zipper H, Brunner H, Bernhagen J, Vitzthum F., Nucleic Acids Res. 2004 Jul 12;32(12):e103.
Bye.
When you add SYBR Green I to your PCR reactions, you will need to add more magnesium. Typically, an increase from 1.5 mM to 4 mM magnesium is required for 1x SYBR Green I, but it may be ueful to titrate in the amount your reactions need.
It is written on a SG I box that 1:1000 of 10000X (thus 10X) give 0.56 optical density at 494 nm. It should be about 10^-5M. Thus X is about 10^-6 M.
When you add SYBR Green I to your PCR reactions, you will need to add more magnesium. Typically, an increase from 1.5 mM to 4 mM magnesium is required for 1x SYBR Green I, but it may be ueful to titrate in the amount your reactions need.
Why is extra Mg needed? I think SYBR intercalates and fluoresces at 1.5 mM Mg. Of course a PCR generally gives more product when run with higher Mg so SYBR will be brighter. If there's something more to it than that then I'd be very interested in a reply
You will probably know it by now...... but for other users:
I also have the 10.000x stock from Roche: dilute it into a 15x stock with DMSO (=7,5� in 5 ml DMSO) and use 1,25� from this in a 25� PCR reaction. Final concentration is 0,75x.
In some cases it is better to use a pre-made kit, but I do this only when a PCR is not working very well or needs to be optimized. When you run a fully optimized PCR you better use your standard conditions and add sybr GrI, that's really the cheapest. But indeed reoptimize your MGCl2 conditions, conc. needs to be higher (don't know why yet.... )
Technician, LUMC, Netherlands
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