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PhasePrep BAC DNA Kit

Scalable method for isolating large-molecular weight plasmids

Synonym: BAC Kit

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Description

General description

The PhasePrep BAC DNA Kit offers a scalable and cost-effective method for isolating large-molecular weight plasmids such as Bacterial Artificial Chromosomes (BAC) from recombinant E. coli cultures. The same kit can be used for preparations of four different sizes; sufficient reagents are provided for 300 micro, 180 mini, 30 midi, or 15 maxi preparations. Up to 2,12, 50, or 100 mg of BAC DNA can be recovered from 5, 40, 250, or 500 mL of overnight recombinant E. coli culture, respectively. The purified BAC DNA contains very low levels of endotoxin (≤10 EU/μg DNA).

Recombinant E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis procedure. Nucleic acid is precipitated from the cleared lysate; residual RNA is removed by a short digestion at elevated temperature with an RNase cocktail. Endotoxins and other impurities are removed by simple temperature-mediated extraction and phase separation. Finally the BAC DNA is selectively precipitated from solution.

The recovered BAC DNA is predominantly in its super-coiled form, free of RNA contamination, and ready for immediate use in downstream applications, such as sequencing, restriction digestion, cloning, and PCR.

The PhasePrep BAC DNA Kit offers a scalable and cost-effective method for isolating large-molecular weight plasmids such as Bacterial Artificial Chromosomes (BAC) from recombinant E. coli cultures. The same kit can be used for preparations of four different sizes; sufficient reagents are provided for 300 micro, 180 mini, 30 midi, or 15 maxi preparations. Up to 2,12, 50, or 100 mg of BAC DNA can be recovered from 5, 40, 250, or 500 ml of overnight recombinant E. coli culture, respectively. The purified BAC DNA contains very low levels of endotoxin (£10 EU/mg DNA).

Recombinant E. coli culture is harvested by centrifugation and subjected to a modified alkaline-SDS lysis procedure. Nucleic acid is precipitated from the cleared lysate; residual RNA is removed by a short digestion at elevated temperature with an RNase cocktail. Endotoxins and other impurities are removed by simple temperature-mediated extraction and phase separation. Finally the BAC DNA is selectively precipitated from solution.

Other Notes

The PhasePrep BAC DNA Kit offers a highly scalable, rapid, cost-effective method for isolating high molecular weight plasmids such as Bacterial Artificial Chromosomes (BACs) from recombinant E. coli cultures.

For additional information, please see www.sigma-aldrich.com/genelutehp.

Application

The recovered BAC DNA is predominantly in its super-coiled form, free of RNA contamination, and ready for immediate use in downstream applications, such as sequencing, restriction digestion, cloning, and PCR.

Features and Benefits

• Typical DNA yields of 2-100 μg from 5-500 mL of overnight cultures
• No phenol/chloroform extraction required
• Allows possible micro to maxi preps with the same kit
• No long waits for drip columns

Legal Information

PhasePrep is a trademark of Sigma-Aldrich Co. LLC

Price and Availability


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Safety & Documentation

Safety Information

Symbol 
GHS07  GHS07
Signal word 
Warning
Hazard statements 
Precautionary statements 
RIDADR 
UN 3316 9
Protocols & Articles

Articles

Plasmid DNA Purification Overview

Extraction of macromolecules such as DNA, RNA, and protein is one of the basic methods used in molecular biology. The process of extraction and purification of nucleic acids has evolved from being a ...
Keywords: Centrifugation, Chromatography, Cloning, DNA purification, Digestions, Genetic, Ion Exchange, Molecular biology, Nucleic acid denaturation, Nucleic acid purification, Polymerase chain reaction, Precipitation, Purification, Sequencing, Transcription, transformation

Peer-Reviewed Papers
15

References

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