Estimation of DNA Quantity on an Agarose Gel - (Dec/28/2010 )

To estimate the DNA quantity on an agarose gel stained with EtBr, should I compare the intensity of my band of interest with the standard band of a similar size, or with the standard band of a similar intensity (assuming there is no standard band that is of similar size and intensity).

just use an marker with bands of different size and intensities

regards,
p

pDNA on Wed Dec 29 16:20:21 2010 said:

So with which of the bands of the marker would you recommend that I compare? The one that is of similar size to my band, or the one that is of similar intensity?

best would be to compare similar size and intensity. But this might not be the case always. One would have to load different amounts of DNA ladder (with conc. eg. 2 log DNA - NEB) and compare intensities.

good luck

And be sure to use a good software. Anyway this is a suboptimal approach, though cheap, as it measures only the end-point which can differ in every reaction due to several reasons (such as deactivation of Taq, shortage of nucleotide substrates and primers, etc).
State-of-the-art an much more exact is qPCR, as the here the exponential and therefore quantifiable part of the reaction is used. But it's expensive and you need a real-time machine.
You might try a competitive PCR as alternative.

Thanks peeps!