Question about gel electrophoresis which is used to measure the concentration of DNA?

[MissP.5_25]

There is a total of 8 wells and the the two ends of the wells are not used, meaning that only 6 wells are used in this experiment. The first four wells are filled with markers while the next two wells are are filled with DNA samples. Below are the amount of markers and DNA which are filled in their respective wells (in microL):

M1 : 1
M2 : 3
M3 : 5
M4 : 10
DNA 1 : 3
DNA 2 : 5

This procedure is carried out to measure the concentration of DNA. Now, what I don't understand:
1. what is the function of the markers
2. why are four markers needed
3. how do you know how many microlitre of markers needed in each well (why 1, 3, 5 and 10)
4. why do you need two DNA samples when they're the same DNA, only different amount
5. how do you know how many microlitre of DNA to put in each well (why 3 and 5)

[hypervalent_iodine]

At a complete guess:

 

Markers are used to determine the size of your DNA fragments. You have 4 of differing amounts, which indicates to me that you are using these to generate a standard curve so you can relate their known concentrations with whatever physical trait you are measuring (absorbance, etc.). This allows you to quantify the concentration of DNA in your sample. The two DNA wells are probably just replicates so you can get an average and a more accurate idea of the concentration of your samples.

 

Edit: I didn't see you'd used different concentrations of your DNA. Not sure why that is.

[CharonY]

There are a number of reasons of which hypervalent_iodine pointed a few out. For practical purposes (e.g. for really quantifying DNA concentration) this particular experiment is not useful as you need more data points for proper calibration and there is smear over around lane 5/6, it appears that some of the marker did not make it into the pockets.

Generally if you put different volumes into the pockets (rather than the same in various dilutions) is if you are not sure how much sample you have got, and you just want to make sure that you have at least one sample that gives a decent signal. I assume that these are PCR products and while the yield is often reproducible in the lab, in lab courses it may get iffy.

 

The chosen volumes are pretty much arbitrary and tend to be in the middle range on the capacity of the average gel pockets (also these are easy to pipet volumes).

Edited October 31, 2014 by CharonY

[MissP.5_25]

At a complete guess:

 

Markers are used to determine the size of your DNA fragments. You have 4 of differing amounts, which indicates to me that you are using these to generate a standard curve so you can relate their known concentrations with whatever physical trait you are measuring (absorbance, etc.). This allows you to quantify the concentration of DNA in your sample. The two DNA wells are probably just replicates so you can get an average and a more accurate idea of the concentration of your samples.

 

Edit: I didn't see you'd used different concentrations of your DNA. Not sure why that is.

 

I get it now! Thank you!