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Fluorescence level in real-time - (Apr/29/2008 )

Hi all,

I need urgent help on this topic.
1. How do I access the total fluorescence level emitted by the DNA product?
2. I'd constructed a standard curve of fluorescence level versus RNA concentration. I got a negative equation ie. as the RNA template concentration increases, the fluorescence level decreases. I'm doing one step RT-PCR. What are the possible explanation?

Help!!!!!

-audreyk-

QUOTE (audreyk @ Apr 29 2008, 11:23 AM)
Hi all,

I need urgent help on this topic.
1. How do I access the total fluorescence level emitted by the DNA product?
2. I'd constructed a standard curve of fluorescence level versus RNA concentration. I got a negative equation ie. as the RNA template concentration increases, the fluorescence level decreases. I'm doing one step RT-PCR. What are the possible explanation?

Help!!!!!



You are correct, with the template concentration increasing, Ct value (reflected by fluorescence intensity) will decrease. The equation coefficient is negative.

-larryking-

Are you working on log concentrations?!


QUOTE (audreyk @ Apr 29 2008, 03:23 PM)
Hi all,

I need urgent help on this topic.
1. How do I access the total fluorescence level emitted by the DNA product?
2. I'd constructed a standard curve of fluorescence level versus RNA concentration. I got a negative equation ie. as the RNA template concentration increases, the fluorescence level decreases. I'm doing one step RT-PCR. What are the possible explanation?

Help!!!!!

-kikinho-