I would like to know the opinion of real-time experts about the following:
Do you think it is correct and acceptable for publication, if I optimize a real-time assay using a buffer developed for probes, and I complement it with EvaGreen?
As far as I know, the buffer I supposed to use has higher MgCl2 concentration compared to the one that the company sells with SybrGreen, but otherwise it is basically the same (except for the dye).
Dear Rita
as Long as the results are good you can use this combination. To control for unspecific or amplification at all we often mix it inverse, adding EvaGreen to a probe mix. This works well.
I wisch you success
René
Intercalating dye could have an inhibitory effect on your PCR reaction. Try an dilution series of the dye added to your reaction and compare your quantative curve with a gel to create an optimal balance between signal intensity and pcr yield. Ready to use sybrgreen mixes have been optimized for this. Good luck!
Thank you All!
Emiel: If the curves are fine and I don't have any unspecific product, do I have to optimize for the dye also?
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