5 replies to this topic

[yoshi]

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).

[pDNA]

just use an marker with bands of different size and intensities

regards,
p

[yoshi]

pDNA, on 29 December 2010 - 08:20 AM, said:

just use an marker with bands of different size and intensities

regards,
p

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?

[scolix]

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

[hobglobin]

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.

[yoshi]

Thanks peeps!