Gel electrophoresis quick questions?

[scilearner]

Hello everyone,

 

I have a very poor understanding in this area.

 

1. In gel electrophoresis, let's say I used many DNA fragments.Then I used a filter paper, and used a probe to detect a specific DNA fragment. Then the pictures you see of electrophoresis, is it this filter paper you see, I mean you can not see the position of fragments in the machine right. Also does this filter paper, exactly mimic the positions of fragments in electrophoresis. Please excuse the stupidity.

 

2. Gel electrophoresis can be used to measure the length of the DNA accurately. Ok if have standard pieces of known size you can do this, but I still don't get how this works. I mean how can the distance of a fragment exactly give the size of the molecule. In electrophoresis particles with different sizes can end up at the same place due to other factors like charge and resistance. So even if you have fragments with known size how does this work.

 

Thanks

Edited January 9, 2011 by scilearner

[cypress]

Hello everyone,

 

I have a very poor understanding in this area.

 

1. In gel electrophoresis, let's say I used many DNA fragments.Then I used a filter paper, and used a probe to detect a specific DNA fragment. Then the pictures you see of electrophoresis, is it this filter paper you see, I mean you can not see the position of fragments in the machine right. Also does this filter paper, exactly mimic the positions of fragments in electrophoresis. Please excuse the stupidity.

 

I don't think I understand your question. Gel electrophoresis and filter paper electrophoresis are distinct as far as I know so I don't understand use of a filter paper unless you mean you use the gel process as an initial method to get rough separation and then the filter paper process to more finely separate a grouping of fragments from the gel process. Is this what you mean?

 

2. Gel electrophoresis can be used to measure the length of the DNA accurately. Ok if have standard pieces of known size you can do this, but I still don't get how this works. I mean how can the distance of a fragment exactly give the size of the molecule. In electrophoresis particles with different sizes can end up at the same place due to other factors like charge and resistance. So even if you have fragments with known size how does this work.

 

It is not exact but it is often very accurate. You are correct that there are several factors in play. One significant issue for example is dealing with molecular configurations that can form tertiary shapes due to hydrogen cross bonding. The key to effective and accurate sorting is in performing the process with different solvents and other agents to effect differences in the fragments that allow for correct conclusions when all the results are compared and contrasted.

[CharonY]

1) Are you mixing up gel electrophoresis and Southern hybridization?

 

2) Free electrophoretic migration is dependent on electrophoretic mobility. For DNA it becomes size independent above a certain length (bit above 20, I think). The major difference, however is the use of a gel. The sieving effect has a dependence on the linear size. Due to this dependence, you can relate the migration back to the DNA length.

Edited January 9, 2011 by CharonY

[Mr Skeptic]

For DNA, the charge to mass ratio is essentially the same regardless of the length of the DNA, but the longer one is bigger and has more resistance in the gel. So the shorter ones go quicker/farther. Generally you include standards of known length to account for some of the details of how it happens, but its still not exact because molecules of the same length could take on different shapes with different resistance.

[CharonY]

Actually the mass does not really play a role in the calculation of electrophoretic mobility (think Smoluchowski). There appears to be a MW-dependence on charge density, that may allow for the differences at lower MW, though. M/Z really only is relevant when e.g. looking at movement of charged particles in vacuum.

Edited January 9, 2011 by CharonY