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Problem using evagreen (evagreen vs sybrgreen) - (Jun/18/2013 )

Hello everyone

I would like to know if anyone have wierd results using evagreen from biorad. I have always used sybrgreen and it gave me good results. But we in the lab changed sybr for evagreen, because it was expected that eva would be more sensitive. Sense then i got wierd results, especially in the melting curve. I have some times, good amplification and wierd melting curves. I have tested everything since plastes, selling tape. I have also tried a different qPCR machine. The problem is the evagreen.

Thank you

-Inês Diniz-

I am sure but as I heard that somebody had the same problem in our university,
I think that some qPCR machines are not compatible with evagreen.

we use this machine and works well with evagreen:
http://www.illumina....s/eco-qpcr.ilmn
althogh it is not durable.

-memari-

Hello Memari

Do you use normal evagreen ou Sso Fast evagreen? What do you mean by "although it's not durable"?

Thank you

-Inês Diniz-

we use Sso Fast evagreen.
but I am not sure which kinds of evagreen has been used by that person.

-memari-

Are you doing just quantitative PCR for expression studies or so?
In that case using SYBR alternative is AFAIK pointless, you don't need higher sensitivity for getting a Ct value and for melting curve it's true Evagreen could have higher discrimination ability but that's not really needed anyway, if you use melting just for checking the specifity of your product. It won't give you any additional information.

More sensitive intercalator dyes are usefull for HRM, but they usually affect amplification more in this applications and often need quite an optimization to work satisfactory.

-Trof-