Biochimie, biotechnologies

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

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

Agarose gel preparation


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

DNA size markers

1 kb DNA Ladder

DNA ladder 1 Kb

(for more, see: http://www.neb.com)

لاحظ أن مجموع الأوزان هو حوالي 500 نانوغرام و هذا هو وزن الحمض النووي ADN الذي تم تحميله في هلام الأغاروز
في حالة إخضاع محلول من ADN ذو تركيز غير معروف، يصبح ممكنا التكهن بتركيزه، انطلاقا من كثافة شريط قطعة  ADN ذات قيمة 125 نانوغرام في غياب تقدير التركيز بالمطياف، تبقى هذه الطريقة تقديرية فقط


A number of proprietary plasmids are digested to completion with appropriate restriction enzymes to yield 10 bands suitable for use as molecular weight standards for agarose gel electrophoresis. The digested DNA includes fragments ranging from 0.5-10.0 kilobases (kb). The 3.0 kb fragment has increased intensity to serve as a reference band. The approximate mass of DNA in each of the bands is provided (assuming a 0.5 µg load) for approximating the mass of DNA in comparably intense samples of similar size.


See Practical work on extraction and purification of plant DNA, Cadi Ayyad University, Marrakech, Morocco (class S5)
loading samples

Practical work on DNA extraction and electrophoresis (grade S4), Cadi Ayyad University, Marrakech, Morocco.


Vidéos du laboratoire (extraits)
(ces vidéos existent aussi dans DVD avec livre)
Extraction de l'ADN (DNA) de folioles de palmier dattier (Vidéo:
homogénéisation du materiel végétal, déproteination, précipitation du DNA par l'alcool,...)



Electrophorèse de l'ADN (DNA) sur gel d'agarose ( Préparation des échantillons, dépôt, migration et révélation par Bromure d'Ethidium, ...)


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Faculty of Sciences, Cadi Ayyad University
Marrakech, 40000, Morocco

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DNA electrophoresis