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Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae

Published on: November 17, 2023

Purification and characterization of YACs containing large inserts.

R M Gemmill¹, R Bolin, W M Strauss

¹Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, USA.

Current Protocols in Human Genetics

|April 23, 2008

Summary

This study details methods for characterizing DNA cloned in yeast artificial chromosomes (YACs) and purifying them. These protocols enable analysis of insert DNA size, complexity, and origin, crucial for molecular biology applications.

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Identification of protein complexes with quantitative proteomics in S. cerevisiae

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Published on: March 4, 2009

Area of Science:

Molecular Biology
Genomics
Yeast Genetics

Background:

Yeast artificial chromosomes (YACs) are essential vectors for cloning large DNA fragments.
Characterizing and purifying YACs is critical for downstream applications like mammalian cell transformation.

Purpose of the Study:

To provide standardized protocols for the characterization and purification of YACs.
To enable researchers to analyze cloned insert DNA and prepare pure YAC DNA.

Main Methods:

Southern blotting and partial-digest restriction analysis for YAC characterization.
Gel purification techniques for obtaining pure YAC DNA.
In vivo fragmentation and Southern blotting for detailed YAC analysis.

Main Results:

Established methods for determining YAC insert size, complexity, and DNA sequence origins.
Provided protocols for purifying YAC DNA suitable for transformation and subcloning.
Demonstrated in vivo fragmentation for advanced YAC structural analysis.

Conclusions:

The described protocols offer robust methods for YAC DNA characterization and purification.
These techniques are vital for successful manipulation and application of large DNA inserts cloned in YACs.