how to estimate amount of DNA using gel? - do u use agarose or acrylamide? (Jan/25/2006 )
hello again...i want to estimate DNA amount by the gel using HindIII marker....I am unsure about the marker concentration but I think 0.25ug/ul.....so in 5 ul which i usually apply I will have 1ug i guess....so can I just take 1ul of my sample put around 0.5ul of loading buffer and run in 1% agarose? and then estimate the intensity of the band comparing to the band of the marker?
If you are not sure about the conc of marker; do not bother - it's waste of time.
Get a marker of known conc. (measure the conc of your marker, or buy a new tube).
Then make a dilution series, or, for a DNA ladder;
calculate the conc of a reasonable number of the bands.
Make sure there will be bands with conc around the conc you expect your sample to have.
Personally I would spend an extra lane for a 10 fold dilution of the marker.
Some suppliers actually market DNA ladders with conc of each band given, so you do not have to do the maths. I used a set from Fermentas, but I guess by now most suppliers have them.
neb and promega give know mass-molecular weight markers too.
i would use agarose gel.