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Related Concept Videos

DNA Agarose Gel Electrophoresis02:35

DNA Agarose Gel Electrophoresis

Agarose gel electrophoresis is a laboratory technique commonly used to separate DNA fragments by size. However, it can also be used to isolate and purify DNA fragments using a gel extraction protocol.
Gel extraction follows five major steps: running gel electrophoresis to separate fragments, isolating the individual bands, extracting DNA from those bands, and removing the dye and salts from the extracted mixture to obtain pure DNA.
In cloning experiments, both the insert and vector DNA...
Two-dimensional Gel Electrophoresis01:22

Two-dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis is a high-resolution protein separation method first introduced by O' Farrell and Klose in 1975. This method involves protein separation by two dimensions, mass and charge, making it more accurate than one-dimensional gel electrophoresis.
The first dimension separation uses the isoelectric focusing or IEF technique performed on immobilized pH gradient (IPG) strips that separate proteins according to their isoelectric points.
Biological samples, such as  cells...
SDS-PAGE01:27

SDS-PAGE

Gel electrophoresis is a method that separates biological macromolecules like nucleic acids or proteins by forcing them to pass through a gel matrix under an electric field.
A variation of gel electrophoresis, termed  polyacrylamide gel electrophoresis (PAGE), is commonly used for separating proteins according to their molecular size by passing them through a polyacrylamide gel. Because of the varying charges associated with amino acid side chains, PAGE can be used to separate intact proteins...
Southern Blot02:57

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Electrophoresis: Overview01:20

Electrophoresis: Overview

Electrophoresis is a powerful analytical separation technique that relies on the differential migration of charged species when subjected to an electric field. The core strength of electrophoresis lies in its ability to separate high-molecular-weight species in complex mixtures. It has found widespread use in biochemistry, molecular biology, and analytical chemistry, allowing the separation of compounds like amino acids, nucleotides, carbohydrates, and proteins with excellent resolution.
There...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

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Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices
09:19

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Published on: March 26, 2018

Denaturing gel electrophoresis for sequencing.

B E Slatko1, L M Albright

  • 1New England Biolabs, Beverly, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary
This summary is machine-generated.

Accurate DNA sequencing relies on high-resolution denaturing polyacrylamide gels. This guide details gel setup, electrophoresis, and processing for optimal DNA sequence determination.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • DNA sequence determination accuracy is critical for genetic research.
  • High-resolution separation of DNA fragments is essential for accurate sequencing.
  • Denaturing polyacrylamide gels are a standard method for DNA fragment separation.

Purpose of the Study:

  • To provide a detailed protocol for setting up, running, and processing denaturing polyacrylamide gels for DNA sequencing.
  • To describe modifications that enhance the length of readable sequence information.
  • To present methods for overcoming gel compressions caused by DNA secondary structures.

Main Methods:

  • Preparation of 40-cm long, uniform thickness denaturing polyacrylamide gels (4-8% acrylamide, 7 M urea).
  • Electrophoresis techniques for DNA sequencing.
  • Processing of gels post-electrophoresis.
  • Gel modifications including wedge-shaped spacers, buffer gradients, electrolyte gradients, acrylamide step gradients, and formamide inclusion.

Main Results:

  • The described protocols enable accurate DNA sequence determination.
  • Modifications can increase the length of readable sequence data obtained from a single gel.
  • Inclusion of formamide effectively overcomes gel compressions due to secondary structures.

Conclusions:

  • Optimized denaturing polyacrylamide gel electrophoresis is fundamental for accurate DNA sequencing.
  • Various modifications offer strategies to improve sequence read length and resolve compression artifacts.
  • This unit serves as a comprehensive resource for DNA sequencing gel protocols.