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Data acquisition for qPCR - Endpoint of annealing or endpoint of amplification? (Mar/16/2010 )

Hi,
I have worked in qPCR with the Light Cycler System with Sybr Green. The acquisition of fluorescence was made at the end of the amplification segment, at the moment that all the DNA is double stranded. Now, I am going to use a Mx3000 equipment and the software MxPro suggest a default thermal profile with acquisition of fluorescence at the end of the annealing segment. Why is that? At that moment only the primers annealed with template are double stranded, and the fluorescence is lower than that detectable at the end of the amplification segment.
I would appreciate your help to understand this.
Thanks!
anacm

-anacm-

anacm on Mar 17 2010, 02:09 AM said:

Hi,
I have worked in qPCR with the Light Cycler System with Sybr Green. The acquisition of fluorescence was made at the end of the amplification segment, at the moment that all the DNA is double stranded. Now, I am going to use a Mx3000 equipment and the software MxPro suggest a default thermal profile with acquisition of fluorescence at the end of the annealing segment. Why is that? At that moment only the primers annealed with template are double stranded, and the fluorescence is lower than that detectable at the end of the amplification segment.
I would appreciate your help to understand this.
Thanks!
anacm


I think u are talking abt a melt curve after the analysis i.e the amplification run... the fluorecence signal will be low as the temerature increases as the double stranded DNA will be converted to singe..The Tm will help u validate if u have amplified your amplicon of interest or not!!!
Hope this helps if i understood it right!!

Best luck

-Pradeep Iyer-

Hi,
Thank you for your answer. I am not asking about the melting curve, maybe my question was clear. Please look at the thermal profiles in the file!
Attached File

-anacm-

anacm on Mar 22 2010, 02:42 PM said:

Hi,
Thank you for your answer. I am not asking about the melting curve, maybe my question was clear. Please look at the thermal profiles in the file!


we had a similar problem when changing from Applied to Light Cycler.

Applied machine used a 2-step programme, annealing and amplification done at 60 degrees (1 minute) with detection at the end.

Light Cycler used 3 step.....

Maybe MxPro wants the same? Im not familiar with this.

-Mullet-