High baseline/noise probes - (Aug/03/2008 )

Hi,

I've just started using some dual labelled oligo probes (FAM/HEX with BHQ1)

In every run I seem to have a highbaseline in the first 0-8 cycles.., my actual probe floursence isn't much higher in the amplification stage than this baseline but the curves are the correct shape.

I'm guessing I can't ignore this intial noise ? Does anyone have any suggestions on how to reduce intial floursence (Is that what it is) in the first 0-8 cycles ?

Thanks

Spoon.

Hi

Its a biorad icycler with IQ Supermix 250nm probe and 500nm primer seems to give me the best signal with an annealing temp of 56'C in a 25ul total volume- I've run a gradient and varied primer and probe concs this seems to be the best. I've not had floursence high than this.

I also use dynamic well factors which inserts an extra 1min cycle at 95'C before the hotstart activation (3mins at 95'C as per instructions of mix).

My intial concern is that total flourescence is very low only hitting about 240rfu. But there is a very high baseline in the initial cycles.
The signal to noise ratio seems very low.

Any thoughts /ideas very welcome .

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