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denaturing polyacrilamide gel

denaturing polyacrilamide gel - RNA Forum

denaturing polyacrilamide gel - RNA Forum. Discuss RNA including mRNA structure and function, rRNA, tRNAs, and the biochemistry of other RNA molecules.


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Old 04-20-2010, 09:11 AM
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Default denaturing polyacrilamide gel



To visualize the results (about 400 and 600 nt) of 2 primer extension reactions on a RNA template I must use a denaturing polyacrilamide gel. Why? Why polyacrilamide and why denaturing?
Besides, I was told that loading the samples is very difficult. whatś the problem?
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Old 10-19-2010, 03:40 AM
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Default Re: denaturing polyacrilamide gel

the reason why you have to use polyacrilamide gel instead of agarose, that's because the cDNA you produced are usually small, and to identify the length of the cDNA is important to use more defined gels;
Denaturing is because you only want the size of the cDNA to take effect not the topology;
The reason why is difficult to load is due to the presence of urea or if you use other denaturant that they will fill the wells and when you load, the samples will flow to the surroundings.
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Old 10-19-2010, 09:39 PM
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Default Re: denaturing polyacrilamide gel

Polyacrilamide gels have three important advantages over agarose gels:

- Better resolution: You can separate and visualize DNA bands with 1 bp of difference (i.e. a 80bp and a 79 bp band).
- Can hold bigger quantities of DNA: You can put up to 10 ug of DNA to a well (of 1 cm x 1 mm) without resolution loss.
- Purity: DNA recovered from this gels is extremely pure and can be used for delicate purposes (like sequencing).

For more info check: Sambrook J, Russell DW. 2001. Molecular Cloning: A Laboratory Manual, Ed. 3, Cold Spring Harbor, NY, US: Cold Spring Harbor Laboratory.
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