Summary of Contents for BIO RAD Aurum Plasmid Mini Kit
- Page 1 Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr. Hercules, CA 94547 USA (510) 741-1000 1-800-424-6723 4110111 Rev C...
- Page 2 ™ Aurum Plasmid Mini Kit Instruction Manual For technical service, call your local Bio-Rad office, or in the US, call 1-800-424-6723...
-
Page 3: Table Of Contents
Section 5 Guidelines for Using the Aurum Plasmid Mini Kit .....3 Section 6 Protocol .......5 Section 7 Troubleshooting Guide . -
Page 4: Introduction
Section 1 Introduction The new Aurum plasmid mini kit is optimized for the purification of up to 20 µg of plasmid DNA, rapidly and inexpensively, without the use of toxic reagents or alcohol precipitations. The use of membranes to bind and purify plasmid DNA, in combination with an optimized column design, minimizes handling and allows plasmid purification to be carried out in either a vacuum or spin format. -
Page 5: Storage Conditions
Microcentrifuge (12,000 x g) Additional equipment required for vacuum format: • Bio-Rad Aurum vacuum manifold with vacuum regulator and column adaptor plate (Cat. #732-6470), or other vacuum manifold with luer fittings (Figure 1). Note: Please read Section 7, Instrument Setup and Use for the Column Adaptor Plate in the Aurum vacuum manifold instruction manual for proper vacuum setup conditions. -
Page 6: Guidelines For Using The Aurum Plasmid Mini Kit
Please read the following guidelines before starting the plasmid purification. Bacterial Growth Guidelines: • The Aurum plasmid mini kit can process cultures grown in a variety of different broths, such as LB (Luria-Bertani broth), LBG (LB + 2% glycerol), SB (Super Broth) and 2x YT. For optimum performance, LB or LBG is recommended for most strains of E. - Page 7 media when 10–12 OD•ml of bacteria are processed per column, although smaller amounts of culture may also be processed. To determine the density of a bacterial culture (OD ), combine 50 µl of bacterial culture with 950 µl growth medium (1:20 dilution). Use the growth medium as a blank and take the spectrophotometric reading at l = 600 nm.
-
Page 8: Protocol
( 12,000 x g) under ambient conditions, with any commercially available microcentrifuge which can accommodate 1.5 ml and 2.0 ml tubes. Please read the section "Guidelines for Using the Aurum Plasmid Mini Kit" before proceeding. Transfer up to 12 OD•ml of plasmid-containing bacterial host to a 1.5–2.0 ml capped microcentrifuge tube (not provided). - Page 9 Aurum mini column Column adaptor plate Manifold top Waste collection tray A stage Manifold base Fig. 3b. Connection of Aurum Fig. 3a. Vacuum setup mini column to column adaptor conditions for the Aurum plate plasmid mini kit Vacuum manifold Vacuum source Filter flask Vaccum regulator Fig.
- Page 10 Note: The neutralization solution should be added within 5 min after lysis. Add 350 µl of neutralization solution and mix by inverting the capped tube briskly 6–8 times. DO NOT VORTEX OR SHAKE. A visible precipitate should form. Centrifuge the neutralized lysate for 5 min. A compact white debris pellet will form along the side or at the bottom of the tube.
- Page 11 Add 50 µl elution solution. Let stand 1 min and then centrifuge 1 min to elute. Purified DNA is ready to use or can be stored at 4°C. Aurum Plasmid Mini Kit: Cat. #732-6400 For more information, call Technical Services at 1-800-424-6723. Bulletin 2663...
- Page 12 Spin Format The Aurum plasmid mini kit can be used with any commercially available microcentrifuge that can accommodate 1.5 ml and 2.0 ml tubes. All centrifugation steps are performed at maximum speed ( 12,000 x g) under ambient conditions. Please read the previous section "Guidelines for Using the Aurum Plasmid Mini Kit"...
- Page 13 1 min to elute. Purified DNA is ready to use or can be stored at 4°C. Aurum Plasmid Mini Kit: Cat. #732-6400 For more information, call Technical Services at 1-800-424-6723. Fig. 6. Aurum plasmid mini protocol overview: spin format...
-
Page 14: Troubleshooting Guide
Section 7 Troubleshooting Guide Problem Possible Cause Possible Solution Low plasmid yields Low copy number Use high copy number plasmid constructs Poor plasmid Inoculate large-scale propagation in culture cultures with overnight cultures generated from fresh colonies grown on a selective medium Determine optimum growth and plasmid propagation times for... - Page 15 Problem Possible Cause Possible Solution Bacterial DNA Excessive amount of Determine OD contamination bacteria processed culture and do not exceed 12 OD•ml of bacteria processed Excessive agitation of Do not shake or vortex lysate lysate after addition of lysis solution RNA contamination Excessive amount of Determine OD...
-
Page 16: Ordering Information
4 replacement luer caps, A and B stages, waste collection tray, vacuum regulator and gauge, tubing, protocol overview, and instruction manual 732-6400 Aurum Plasmid Mini Kit, 100 preps, includes 100 plasmid binding columns, 100 capless collection tubes (2.0 ml), reagents, protocol overview, and instruction manual...