Description
General description
Click here to view the kit protocol
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.
Application
PhasePrep™ BAC DNA Kit has been used to isolate bacterial artificial chromosomes.
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
Typical DNA yields of 2-100 μg from 5-500 mL of overnight cultures
Allows possible micro to maxi preps with the same kit
No long waits for drip columns
Other Notes
For additional information, please see www.sigma-aldrich.com/genelutehp.
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.
Legal Information
PhasePrep is a trademark of Sigma-Aldrich Co. LLC
