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DNA. Separation by electrophoresis on agarose gel
تنقية الحمض النووي و التحليل  بالكهرتهجير


DNA separation could be carried out using:

- Electrophoresis on agarose gel
- Electrophoresis on polyacrylamide gel

Electrophoresis separates linear double strand DNA respecting its size. For single strand DNA and RNA, denaturant buffer (plus urea) must be used because of the secondary and tertiary structures not allowing size-based separation.

Electrophoresis on agarose gel

Agarose gel electrophoresis separates nucleic acids on the basis of their electric charge (-). Small molecules move fast and show high migration, when compared with molecules of high size.

Agarose gel

Efficient range of separation of agarose gel using a double strand linear DNA

Percentage of agarose in gel (%) Efficient range of separation (bp)

0.3 ......
0.6 ......
0.7 ......
0.9 ......
1.2 .....
1.5 .....
2.0 ......

5000-60000
1000-20000
800-10000
500-7000
400-6000
200-3000
100-1200

- Highly concentrated gels are suitable for separation of small DNA fragments.
- Low concentrated gels are suitable for separation of big DNA fragments.

Applications

- Determination of molecular weight of DNA fragments after digestion by restriction enzymes
- DNA and RNA after their amplification by PCR and RT-PCR.
- Separation of DNA fragments before Southern blotting and RNA fragments before Northern blotting.

Separation of plasmids by agarose electrophoresis.

Plasmids, such as pBr 322, exist under 3 forms depending on their conservation statute:

- Covalently closed circular (CCC) or supercoiled (Forme superenroulée in French). This form occurs when the plasmid solution is well conserved
- Open circular (OC) (Forme ouvert circulaire ou forme relâchée, in French). This form occurs when plasmid is not well conserved.
- Linear plasmid (Forme linéaire, in French). This form is obtained when plasmid is digested by restriction enzymes

We can obtain the two first forms at the same time. In this case, more bands will be observed on agarose gel.

DNA conformation affects electrophoretic migration. To exclude this effect when the objective is to determine molecular weight, only linear DNA (result from digestion, amplification by PCR, ..) is subjected to electrophoresis.
Non digested plasmid DNA, migrates at different rates.

plasmid. 3 forms
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