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Using QPCR and Sybr I to measure oligo probe Tm?


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#1 grkn

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Posted 30 December 2010 - 02:53 PM

I'm investigating the possibility of making Tm measurements using short 9-20mer oligos targeting e.g. ssLambda and measuring Sybr I fluorescence from 90C to 4C to determine the probe Tm - when the temperature is lowered from 90C probe you see more hybridization and Sybr I emission should increase due to the increase of dsDNA (according to invitrogen dsDNA generates ~10x more signal than ssDNA).

How sensitive are QPCR machines compared to fluorescent plate readers using Sybr I and looking for dsDNA? i.e. how many ng of Sybr I stained dsDNA is A ) detectable and B ) differentiable/with what resolution? We have an old plate reader that can detect 1ng of Sybr I stainted Lambda in 100ul solution, with a resolution of about 1-2ng.
I see that many QPCRs claim to be able to resolve 5000 from 10000 copies of a ~350 nucleotide RNA, which would amount to attograms of material?

Would it also be possible to make this (semi) quantitative by using know amounts of dsDNA as a standard?

Cheers,
John

Edited by grkn, 30 December 2010 - 03:00 PM.





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