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Electrophoresis samples and volumes


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#1 Biologystudent

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Posted 29 March 2014 - 02:37 AM

Question 1)

Is it necessary to add water to your samples when running electrophoresis? I mean, does water have a specific purpose or is it just to add volume?

Usually for 5ul of DNA-samples I go with the following:

- 5ul DNA sample

- 5ul water

- 2ul loading dye

_____

Total of 12microliter to load into the well.

 

 

Question 2)

But when I have bigger volumes, 45-50ul. Then I just go with the following:

- the DNA-sample (45-50ul)

- No water at all

- plus 1/6th loading dye of the total reaction volume

 

Does this sound correct with adding loading dye in a  1/6th-ratio of the total reaction volume?

And having no water at all? I don't get why water is good in the first instance, but can be skipped in the other instance.

 

Question 3)

I want to run electrophoresis with a 50ul DNA-sample to see whether the digestion was successful (gave me 2 separata bands on the agaros).

If I then want to purify the DNA from the excised agaros-bands afterwards, would you recommend to either:

- load the entire 50ul sample into 1 single agaros-well?

- or divide the sample into separate lanes (for instance 5wells/10ul alternative 2wells/25ul each)?

 

I prefer to divide the whole sample into separate wells, rather than just 1 big well for the entire sample. Would this be bad?

 

Question 4)

When running the whole sample in a big well, I use a comb that generates a well in the agaros that has the same depth as regular wells, but just is more extended in horisontal orientation (so the whole sample can fit). Would the wells dimension affect the migration in any way?


Edited by Biologystudent, 29 March 2014 - 02:38 AM.


#2 jerryshelly1

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Posted 29 March 2014 - 11:54 AM

1) I never add water. Are you adding water to get your DNA dye down to 1X? When I set up my PCR I add a consistent concentration of DNA so I can quantify my amplicons using the DNA ladder.

 

2) You probably don't add water here because you would overload the well and your sample mix would spill out. 

 

3) Use one lane if the well can accommodate the entire sample. This will reduce the total amount of agarose an reduce the volume of reagents that will be required for gel purification.

 

4) No. Review the methodology behind electrophoresis on this website or wiki.






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