DNA quantitation on agarose gel - (Sep/17/2006 )
Hi,
How do you determine concentration of your DNA sample by comparing with the DNA ladder? 
Thank you.
Hi zfin,
to determine the concentration of DNA you first check which band of the ladder (which concentrations you know) is most similar with your DNA sample.
Now you have to calculate:
The amount of the ladder band is given, for example 80ng (it depends on the volume you applied...). So your DNA has ~40ng/(volume of the sample you applied).
Hope it's clear...?
Greetings,
Chakchel
creating a simple standard curve is also possible (known concentrations against the distance each band migrated)
I don't see how that would work. Concentration has nothing to do with the distance each band has migrated. Migration distance has more to do with band size and gel density. You would find the floresence intensity of a band is dependent on its lenght and concentration. Thus from floresence intensity one can work out concentration if the band size is known.
my apology..perneseblue.........i mean distance VS size
though u make me more confused
I agree with perneseblue.
One compares intensity of a DNA band(known conc.) to a DNA band of unknown conc..
i am totally agreed with the method of both of u ...
but we use a standard curve for determination of unknown proteins..is this also wrong...i'm getting confused
but we use a standard curve for determination of unknown proteins..is this also wrong...i'm getting confused
No, no, it should be right. A (distance vs size) standard curve... is okay as long as 2 criterior are met.
1 - the protein is denatured, and not heavily glycosylated or charged/phosphorylated
2- the gel density used is always the same.
All I was saying in my previous post in regrads to migration distance, was that it depended on both the size (and shape in native protein gel) and the gel density.
I'm sorry for having caused a panic.
hi zfin,
I sort of agree with the comments but make sure that the the ref DNA (ladder) and the sample is in the same solution and both in the same confugaration (denatured or not denatured). Secondary or tertiair sturctures of you DNA can cause concentration problems compared to the ladder.
I prefer to just measure the OD 260 with a spectofotometer (e.g.nanodrop)
good luck
You get quantifiable DNA ladders. (Eg)
Quantity of DNA in each band (100bp, 200bp.....) are given. So compare the intensity of youe sample DNA band with the ladder band. Though it is not very accurate, works fine for most purposes.