DNA separation could be carried out using:
- DNA electrophoresis on agarose gel
- DNA 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.
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
...... |
5000-60000 |
-
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.
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.
| DNA
size markers
1 kb DNA Ladder 
(for more, see: https://www.neb.com) لاحظ
أن مجموع الأوزان هو حوالي 500 نانوغرام
و هذا هو وزن الحمض النووي ADN الذي تم
تحميله في هلام الأغاروز |
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) |
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, ...)
 |
 |
 |
Liens utiles:
- Extraction et
purification de l'ADN des plantes (TP S5)
- DNA electrophoresis
- Electrophorèse de l'ADN
des plantes (TP S5). QCM
- QCM électrophorèse,
supports de migration
- QCM Chromatographie,
Electrophorèse
- QCM Nucléosides
& Nucléotides
- Purification, analyse DNA
du plasmide. Contrôle TP 2015
- Biologie
moléculaire. Examen TP S5 2016
- Contrôle (Examen)
de TP Biologie moléculaire, 2019
- DNA electrophoresis on agarose gel
- DNA electrophoresis on agarose gel step by step
- Clonage
des gènes et génie génétique
Video 'genetic diversity through molecular markers' (master level, Cadi Ayyad University, 2019:
-
تحميل ملفات في مواضيع البيوكيمياء
Téléchargement de fichiers en Biochimie
Chaine
Youtube (abonnement). Plusieurs vidéos multilingues
Congrès, Conférences,...
- Congrès, Conférences
- Cours, Workshops
- Bourses, Postdocs
- Biotech-ecolo annonces
Afin de pouvoir continuer à servir les visiteurs, soutenez nos actions sur le site takween en faisant acquisition des ouvrages et supports pédagogiques desitinés à améliorer l'enseignement et la recherche scientfique en Biochimie.
Faculty of Sciences, Cadi Ayyad University
Marrakech, 40000, Morocco
Email: baaziz@uca.ac.ma
Phone: 212524434649 (post 513)
Fax: 212524434669
About takween.com
