How to calculate DNA concentration after electrophoresis - hello (May/11/2007 )
can u help me ,how to calculate the concentration????
we have a standard picture of gel electorphoresis of serial dilution of DNA
it starts from 5ng, 10 ng, 50 ng, 100 ng, 200 ng, 500ng, 1 ug etc
so when we run our sample in a gel and after take its picture we compare the band of our sample with the standard picture , and we assume a rough extimation of DNA by this way
Even with the standard picture, you have to look at the size. Different size has different intensity.
It is really rough estimation. I used the DNA marker instead. Easier that way. I usually wouldnt use it to calculate.
Spectroscopy and of course Nanodrop will be the best option.
Why? Do you not have a spec? You should measure absorbance of 260 and calculate the from that. Eventually if you have done a lot of molecular biology you will get better at guessing at the concentration from the gel. But IMO if you have little experience this is not a good method. For example, a 10kb fragment will be much more brighter than a 1kb fragment. So, you must compare to serially diluted DNA ladder and you must compare to fragments of approximately the same size.
Which DNA ladder do you usually use?. Lamba DNA or LamDNA/HinIII for instance?. Which one best?
Lambda hind3 is a good ladder for DNA estimation.
We use 2log DNA (NEB), which has smaller DNA fragments with specific concentrations for each band.