NA0100

PhasePrep^™ BAC DNA Kit

Scalable method for isolating large-molecular weight plasmids

• Synonym(s): BAC Kit
• NACRES: NA.52
• UNSPSC Code: 41105501

Properties

• Quality Segment: 200
• technique(s): DNA extraction: suitable
• storage temp.: 15-25°C

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

Kit Components Also Available Separately

• R6148

  RNase A solution 2

  SDS
• R1149

  Resuspension Solution, Add RNase A before use. After addition of RNAse, store at 2-8ºC. ) 2

  SDS
• L1912

  Lysis Solution 2

  SDS
• E4274

  Endotoxin Removal solution, Remove lipopolysaccharides (LPS) from plasmid DNA preparations

  SDS
• S2404

  Sodium acetate buffer solution, pH 7.0±0.05 (25 °C), BioXtra, Molecular Biology, 3 M, non-sterile; 0.2 μm filtered

  SDS

Safety Information

• Pictograms: GHS08,GHS05,GHS07
• Signal Word: Danger
• Hazard Codes: H290,H315,H319,H334,H336
• Precautionary Statements: P234 - P261 - P264 - P271 - P302 + P352 - P305 + P351 + P338
• Hazard Classifications: Eye Irrit. 2 - Met. Corr. 1 - Resp. Sens. 1 - Skin Irrit. 2 - STOT SE 3
• Target Organs: Central nervous system
• Flash Point (°F): Not applicable
• Flash Point (°C): Not applicable
• WGK: WGK 3
• Storage Class: 10 - Combustible liquids