Electrophoresis of DNA (80-200 base pairs)

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Bronwen Dekker

Friday, 20 Apr 2012 09:26 UTC

A researcher has emailed with some questions, which I think are:

Regarding: Electrophoresis of DNA (80-200 base pairs)

Should you do electrophoresis using agarose or polyacrylamide?

What length of gel is required?

Thank you for your help with this!

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Copied email:

Hi, I am researcher on a some DNA markers that amplification by PCR, I want they electrophoresis according their sizes that about (80-200pb).What length a gel is require ?
and DNA ladder fragments? And the elecrophoresis is best in agarose or polyacrylamide?
Please, you answer me about these .
Thank you very much

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Anonymous

21 Apr 2012 | 21:55

Hi there,

In my case, my DNA samples are around 150 to 240. 
I use 2% agarose gel. 100V for 20min to 30min.

And you can check this link as well. 
http://gcat.davidson.edu/iGEM08/gelwebsite/gelwebsite.html

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Harshad Mayekar

22 Apr 2012 | 06:01

the porosity of the gel is determined by the DNA you are going to load. Accoriding to your needs, I think 2% of agarose gel is a feasible option. Polyacrylamide is for proteins which are weighed in Daltons or KiloDalton. For DNA we use agarose gels, because DNA is a huge entity.

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Anichavezhi,D Devendran

27 Apr 2012 | 06:39

1% or 2 % of agarose would just be fine as per your requirement of resolution check is concerned (120 V for 30 min. And as per the above mentioned reply, the porosity of polyacrlamide gel is less when compared to the agarose gel, and henceforth used to check the resolution of smaller nucleic acids like restriction digested products or proteins.

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Bronwen Dekker

27 Apr 2012 | 16:33

Thank you for the comments!

I am still not quite sure what the recommended length for the gel is. Would 10 cm be okay?

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Harshad Mayekar

28 Apr 2012 | 09:32

Regarding the doubt on gel length. Do you want to know about the length od the caster of the apparatus. Then yes, I think 10 cm of the gel length is sufficient. If you get one of the gel kits i think the length is around 10-15 cm. If you are trying out DNA, certainly this much length is sufficient. You also get casts which have dents at two places, such that you can place two combs; one after the other and run twice as many samples. The lengths in such cases is lesser than 10 cm. Does that answer the question?

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Bronwen Dekker

30 Apr 2012 | 20:46

Thank you, Harshad; that is very helpful!

Scrolling through the internet, I found out that the length of the gel researchers used for this type of experiment was somewhat difficult to pin down. In my travels I came across the idea that Small 8×10 gels (minigels) are very popular and give good photographs. And: if, for example, you were measuring telomere length by the Southern blot analysis of terminal restriction fragment lengths, you would use 0.5% (wt/vol) agarose to make a 20 cm × 20 cm × 1 cm thickness gel for standard leukocyte telomere length measurements (3–20 kb).

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Harshad Mayekar

01 May 2012 | 16:05

It is my pleasure if I have been of little help if any, Bronwen. All the best for your gel running!

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B Ping

04 May 2012 | 15:00

Hi

We use polyacrylamide gels at work on a regular basis (TBE Gels 6%) with very good results. I think it all depends on the resolution that you want to achieve with your fragments. We used to prepare our own gels but we have had much better results since we started using pre-cast gels. If it is a case of just seeing the bands vs. 2-3 bp resolution I would approach this differently…