Gel Electrophoresis Question - (Nov/26/2010 )
HomeBrew on Sat Nov 27 02:50:31 2010 said:
You can even go a little further, if you want (this sounds like a school experiment, so I'm going to assume you're a student -- no offense meant if you're not...). The relationship between the brightness of ethidium bromide stained DNA is directly proportional to the amount of DNA present. So, if you look at lane seven, the smear you can see in the lower part of the gel (smaller fragments) is about 1/4 - 1/2 as bright as the ladder in that region. So, if you know the amount of DNA that's present in the ladder in that region, you can estimate how much DNA there was in lane seven for fragments of that size.
To do this, would it simply be a ratio? I am confused as to how to figure this out because I'm not sure what to compare to. The bands that appeared were from the ladder which obviously don't contain DNA but the same amount of ladder and sample were loaded into the wells. How would we go about the calculations?
The ladder is composed of DNA. Some types of ladder have specifications on how much DNA is contained in each band, so you can do a rough quantitation off the relative brightness of the bands. Ask your lab demonstrators which brand and the name of the DNA ladder (my guess is 100 bp marker from Invitrogen), then you can look up the ladder on the companies' website and see if there are quantitative bands.
The ladder *is* DNA. It was purchased from some company at a certain concentration. Let's say I was using 1 Kb Plus DNA Ladder from Invitrogen (see here). This ladder is sold as 250 μg at a concentration of 1 μg/μl.
Let's say I loaded 10 μl of this ladder on my gel -- if so, then I've loaded 10 μg of DNA in that lane (waaayyy too much, but let's just continue).
Invitrogen's literature on this ladder says that "the 1650-bp band contains approximately 8% of the mass applied to the gel". If so, then the 1650-bp band contains 0.8 μg of DNA (or ~0.74 picomoles of DNA for a 1650-bp piece).
If I look at the brightness of the 1650-bp band in the ladder, and compare it to a band on my gel, and it's about the same, then my sample band is present at ~0.74 picomoles; if it's twice as bright, then there's about 1.48 picomoles, etc.