I am a first year science undergraduate and I am just asking this as a more theoretical question rather than for carrying out a protocol, so I would appreciate if answers do not involve lots of complicated chemical names.
I was wondering how one would go about quantifying the amount of DNA in a given band on a gel electrophoresis. Some ideas I have had so far are:
- Cut out the DNA band and extract the DNA. Quantify using some known method such as measuring the absorbance at 260nm of the DNA in solution to find the concentration.
- When the DNA is stained with ethidium bromide, clearly there will generally be more stain uptaken in a band region if it has more DNA so it will fluoresce with greater intensity when illuminated with UV light. However I would have reservations to using this method because I would think that the amount of stain uptaken would depend also on the state of the DNA (i.e. if it is supercoiled or not), but then again I do not know how much of an impact this could have on how much stain is uptaken. Of course, if you know that all of your DNA is in the same state then there shouldn't be a problem with this method I think. Although here you would have to be aware that the concentration of ethidium bromide in a certain band depends on the concentration of nucleotides there, which depends both on the number of the DNA fragments and their size (which is known from the position of the band). Compare this with the final method...
- Finally, if one is making the DNA for example using dideoxy chain termination/Sanger sequencing in PCR, then you can use a radioactively labelled primer. The intensity of the image on the radioautograph would then directly tell you the number of molceules of that DNA fragment present in the band.
I was wondering which of these are viable options to use in the lab, and why/why not.