PhasePrep™ BAC DNA Kit
SIGMA/NA0100 - Scalable method for isolating large-molecular weight plasmids
Synonym: BAC Kit
Product Type: Chemical
BAC DNA was purified with the PhasePrep™ BAC DNA Kit and sequenced with a custom primer using BigDye® Terminator Chemistry.
Sequencing reactions were resolved on an ABI 3700. Data provided by the Genome Sequencing Center at Washington University, St. Louis
Sequencing reactions were resolved on an ABI 3700. Data provided by the Genome Sequencing Center at Washington University, St. Louis
Purified BAC DNA is suitable for restriction digestion.
Restriction digestion of BAC DNA isolated PhasePrep™ BAC DNA Kit and two other suppliers (C and Q). BAC DNA samples were purified from overnight cultures of E. coli HB101b transformed with a pbelloBAC11 clone. Approximately 1 μg of DNA from each sample was digested with 20 units of EcoR V at 37 °C for 4 hr. One half of each digestion (approximately 0.5 μg) was separated by overnight electrophoresis in 1% agarose gel. Lanes 4-19 were BAC DNA purified with Sigma kit, lane 1 was BAC DNA purified with supplier C’s kit, and lane 2 was BAC DNA purified with Supplier Q’s kit. The molecular marker used at the first and last lanes was a 1 kb DNA Ladder (Cat. No. D0428).
Restriction digestion of BAC DNA isolated PhasePrep™ BAC DNA Kit and two other suppliers (C and Q). BAC DNA samples were purified from overnight cultures of E. coli HB101b transformed with a pbelloBAC11 clone. Approximately 1 μg of DNA from each sample was digested with 20 units of EcoR V at 37 °C for 4 hr. One half of each digestion (approximately 0.5 μg) was separated by overnight electrophoresis in 1% agarose gel. Lanes 4-19 were BAC DNA purified with Sigma kit, lane 1 was BAC DNA purified with supplier C’s kit, and lane 2 was BAC DNA purified with Supplier Q’s kit. The molecular marker used at the first and last lanes was a 1 kb DNA Ladder (Cat. No. D0428).
Figure 1: BAC DNA was purified with the PhasePrep™ BAC DNA Kit and sequenced with a custom primer using BigDye™ Terminator Chemistry. Sequencing reactions were resolved on an ABI 3700.Data provided by the Genome Sequencing Center at Washington University, St. Louis
| Quality Level | 200  |
| storage temp. | 15-25°C |
| technique(s) | DNA extraction: suitable |
| Application: | PhasePrep™ BAC DNA Kit has been used to isolate bacterial artificial chromosomes. |
| 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 • Allows possible micro to maxi preps with the same kit • No long waits for drip columns |
| 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 |
| General description: | Click here to view the kit protocol |
| 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. |
| 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/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. |
| Legal Information: | PhasePrep is a trademark of Sigma-Aldrich Co. LLC |
| Other Notes: | For additional information, please see www.sigma-aldrich.com/gen. |
| 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. |
| Symbol |    GHS05,GHS07,GHS08 |
| Signal word | Danger |
| Hazard statements | H290 - H315 - H319 - H334 - H336 |
| Precautionary statements | P234 - P261 - P264 - P271 - P302 + P352 - P305 + P351 + P338 |
| Hazard Codes | Xi |
| Risk Statements | 36/38-67 |
| Safety Statements | 26 |
| RIDADR | UN 3316 9 |
| Flash Point(F) | Not applicable |
| Flash Point(C) | Not applicable |
| Storage Temp. | 15-25°C |
| UNSPSC | 41105501 |
| Components | RNase A solution 2; Resuspension Solution, Add RNase A before use. After addition of RNAse, store at 2-8ºC. ) 2; Lysis Solution 2; Endotoxin Removal solution, Remove lipopolysaccharides (LPS) from plasmid DNA preparations; Sodium acetate buffer solution, pH 7.0±0.05 (25 °C), BioXtra, Molecular Biology, 3 M, non-sterile; 0.2 μm filtered |