Related Experiment Video
Updated: Jul 5, 2026
11:19
Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
Published on: August 21, 2016
Pulsed-field gel electrophoresis for long-range restriction mapping
Robert M Gemmill1, Richard Bolin, Hans Albertsen
1Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, USA.
Current Protocols in Human Genetics
|April 23, 2008
Summary
This study details methods for creating long-range restriction maps of genomic DNA and analyzing large DNA fragments. It covers DNA digestion, separation, and hybridization techniques for genomic research.
Area of Science:
- Genomics
- Molecular Biology
- Biotechnology
Background:
- Generating accurate long-range restriction maps is crucial for understanding genome structure and function.
- Analyzing large DNA inserts, such as those in Bacterial Artificial Chromosomes (BACs), presents unique technical challenges.
Purpose of the Study:
- To provide a comprehensive set of protocols for generating long-range restriction maps of genomic DNA.
- To detail methods for the analysis of large insert clones, including BAC DNA preparation.
Main Methods:
- Restriction digestion of agarose-embedded genomic DNA.
- Pulsed-field gel electrophoresis (PFGE) for separating large DNA fragments.
- Southern transfer and hybridization techniques.
- Preparation of high-molecular-weight DNA in agarose blocks and microbeads.
- Development of DNA size standards from lambda phage and yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe).
- Alternative method for yeast DNA size standard preparation using lithium dodecyl sulfate (LiDS).
- Protocol for preparing BAC DNA for subsequent analysis.
Main Results:
- Established procedures for restriction digestion, PFGE, Southern transfer, and hybridization.
- Demonstrated methods for preparing high-quality, high-molecular-weight genomic DNA suitable for long-range mapping.
- Provided reliable DNA size standards essential for accurate PFGE analysis.
- Detailed a robust protocol for BAC DNA preparation, enabling downstream applications like mapping and sequencing.
Conclusions:
- The described protocols offer a robust framework for generating long-range restriction maps and analyzing large DNA inserts.
- These methods are essential for comprehensive genomic analysis and the characterization of complex DNA constructs.
- The unit provides valuable tools for researchers working with large-scale genomic data and cloning systems.
Related Concept Videos
01:22Two-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...
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...
02:35DNA 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...
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...
02:57Southern 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...
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...
01:27SDS-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...
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...
01:20Electrophoresis: 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...
There...