Is it possible a DNA with an absorbance ratio A260/A230 = 2.96, for example, be influencing the low fluorescence in the Real-Time PCR? I was trying to optimize a real time PCR method to amplify a specific segment and for this purpose, I was using primers which were designed for a conventional PCR and the fragment lenght is 200bp. Could be this the problem which is causing the low fluorescence?
I've already used different SybrGreen Master Mixes...
Hello,
I think that fragment length is ok.
What is the concentration of your DNA sample and how many µL did you use for your real time PCR ?
Moreover, absorbance ratio A260/A230 of 2.96 sounds a bit high for DNA, no?
Did you use CTAB during your DNA extraction ? If yes, CTAB absorb at 260 nm and can interfer during DNA quantification when using spectrophotometer like Nanodrop.
The concentrations of my samples vary between 30 and 70ng/uL and I'm using 5uL at the real time PCR reactions.
I extracted them with CTAB and I'm using NanoDrop to quantify them.
My doubt relies on the low fluorescence on the real time pcr , which was always about 0.2.
For RT-PCR, I see that you are using too much DNA template, try to adjust your concentration in all the samples to be 20 ng/ul and use for from that just 3-5 ul fro your PCR, remember Rt-PCR is very sensitive so around 50 ng per reaction will be more than enough. Also, try to use another set of primer because I have this experience before.
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