How to extract DNA from polyacrylamide gel electrophoresis?

[Guest]

Hey everybody. I'm a final year student, and I have to do some experiments with SSR (simple sequence repeat) indicators. Now I'm preparing to cloning some genes, and I have some troubles. It's the DNA extraction stage, but from 6% silver- staining polyacrylamide gel (normally, in my lab, they just extract from agarose gel). I need some advices about the protocol and the difference between the ethidium bromide - staining polyacrylamide gel and the silver - staining one which I have to be noticed before I go to extract DNA.

The interested band is about 300 bp and I have to electrophoresis PCR products on polyacrylamide gel because there're many bands which have their size near the band I need (e.g 350bp, 270bp..). Can I use the QIAEX II Gel extraction Kit of QIAgen for this purpose? Other than this Kit, is there any procedure I can use? (this Kit is too expensive, so if there's any cheaper way, it's better),

Please help me, thank for your help!!!

[CharonY]

The qiaex kit contains a protocol for the extraction of DNA from polyacrylamide gels, if I recall correctly. Essentially you need to let the DNA diffuse out of the gel matrix with a certain buffer, then you have separate the gel from the DNA solution (as it cannot be dissolved like agarose gels).

The recovery rate was lower though. You can also do it without columns, but then you need to clean up e.g. by ethanol precipitation. Should work, but the recovery is likely to be even lower.

[DrDNA]

"crush and soak" method of DNA extraction from PAGE gels is relatively simple and usually works well.

http://nationaldiagnostics.com/article_info.php/articles_id/40

[Guest]

"crush and soak" method of DNA extraction from PAGE gels is relatively simple and usually works well.

http://nationaldiagnostics.com/article_info.php/articles_id/40

Thank you for your answer.

[Guest]

Thanks for your all replies.

[Guest]

Why is a stacking gel not required in Agarose gel electrophoresis but needed in Polyacrylamide electrophoresis.

[zule]

Why is a stacking gel not required in Agarose gel electrophoresis but needed in Polyacrylamide electrophoresis.

 

Agarose gels are usually run horizontally, whereas polyacrilamide ones are done vertically.

 

In this way, the samples in the agarose gel are put into wells which are higher than wide. After that, those samples are going to run horizontally, so, all the volume of a sample practically starts “the race” at the same position.

 

In the polyacrilamide gels, wells are also higher than wide, but they are going to run vertically. So, the proteins in the sample which are on the bottom of the well “play with advantage” regarding the ones which are on the surface. The mission of the stacking gel is to concentrate the proteins, so that when the sample arrives at the running gel they are practically in the same line, so the ones on the bottom “don’t play with advantage”. The stacking gel is especially important if there is put a big volume in each well.

[Guest]

Agarose gels are usually run horizontally, whereas polyacrilamide ones are done vertically.

 

In this way, the samples in the agarose gel are put into wells which are higher than wide. After that, those samples are going to run horizontally, so, all the volume of a sample practically starts “the race” at the same position.

 

In the polyacrilamide gels, wells are also higher than wide, but they are going to run vertically. So, the proteins in the sample which are on the bottom of the well “play with advantage” regarding the ones which are on the surface. The mission of the stacking gel is to concentrate the proteins, so that when the sample arrives at the running gel they are practically in the same line, so the ones on the bottom “don’t play with advantage”. The stacking gel is especially important if there is put a big volume in each well.

Thanks for your answer. I got another information is it true. The quick answer is resolution. Agarose gels are typically used for separating big things, like nucleic acids, based on size and the resolution is generally not as important because the molecules are so big. The acrylamide gel has a lot smaller pore size and typically is used for separating smaller molecules (such as small proteins) because it gives you better separation of molecules that are relatively close in size.

 

The stacking gel is important because it allows for all of the proteins to hit the resolving gel at the same time so that in your results you can be sure that the bands at the bottom are actually smaller than those above them, not just that they were just lower in the vertical well and started moving first. The stacking gel is lower in pH and in the pores in this portion of the gel are bigger so there is virtually no separation based on size and all proteins across all wells will start running on the resolving gel at the same time.

 

gel electrophoresis

[Guest]

Thanks for your information.

Biochemical techniques

[Ahamed]

http://globaltutor.blogspot.com/2009/12/how-to-extract-dna-of-cells-of-fruit-by.html