Bio-Rad Laboratories, Inc. 2000 Alfred Nobel Dr.
Aurum™ Plasmid Mini Kit Instruction Manual For technical service, call your local Bio-Rad office, or in the US, call 1-800-424-6723
Table of Contents Section 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Section 2 Kit Components . . . . . . . . . . . . . . . . . . . . . . . . .1 Section 3 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . .2 Section 4 Necessary Supplies . . . . . . . . . . . . . . . . . . . . . .2 Section 5 Guidelines for Using the Aurum Plasmid Mini Kit . . . . . . . . . . . . . . . . . . . . .3 Section 6 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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. In either format, the final elution is carried out in a microcentrifuge.
Section 3 Storage Conditions Solutions and columns should be stored at room temperature. If precipitation is observed in any solution, warm solution to 37ºC to redissolve, and allow to return to room temperature before use. Do not expose any of the solutions to temperatures above 37ºC. If the kit is used infrequently, storage of the resuspension solution at 4ºC is recommended to preserve the RNase. Section 4 Necessary Supplies Equipment and supplies to be provided by the customer: • 1.5–2.
Section 5 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. coli.
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 (OD600), 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. Multiply this figure by 20 to calculate the bacterial concentration.
Fig. 2. Vacuum regulator with clear representation of the gauge Section 6 Protocol Vacuum Format This procedure requires the Bio-Rad Aurum vacuum manifold and column adaptor plate (Cat. #732-6470), or any vacuum manifold with luer fittings. For proper vacuum setup conditions, please read Instrument Setup and Use for the Column Adaptor Plate in the vacuum manifold instruction manual, and see Fig. 4.
Aurum mini column Column adaptor plate Manifold top Waste collection tray A stage Manifold base Fig. 3b. Connection of Aurum mini column to column adaptor plate Fig. 3a. Vacuum setup conditions for the Aurum plasmid mini kit Vacuum manifold Filter flask Vaccum regulator Vacuum source Fig. 4.
Note: The neutralization solution should be added within 5 min after lysis. 4. 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. 5. Centrifuge the neutralized lysate for 5 min. A compact white debris pellet will form along the side or at the bottom of the tube. 6.
Aurum Plasmid Mini Kit: Vacuum Format ™ ™ Protocol Overview For complete protocol, consult instruction manual. Growth and Isolation 1. Grow 1–5 ml bacterial culture overnight or 16 hr. 2. Measure A600 (if higher yield required). 3. Transfer an appropriate volume of culture to a capped 2 ml tube. Centrifuge and decant supernatant. 4. Add 250 µl resuspension solution; vortex. 5. Add 250 µl lysis solution; invert 6–8x. 6. Add 350 µl neutralization solution; invert 6–8x. 7.
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" before proceeding. 1. Transfer up to 12 OD•ml of plasmid-containing bacterial host to a 1.5–2.0 ml capped microcentrifuge tube (not provided).
Aurum Plasmid Mini Kit: Spin Format ™ ™ Protocol Overview For complete protocol, consult instruction manual. Growth and Isolation 1. Grow 1–5 ml bacterial culture overnight or 16 hr. 2. Measure A600 (if higher yield required). 3. Transfer an appropriate volume of culture to a capped 2 ml tube. Centrifuge and decant supernatant. 4. Add 250 µl resuspension solution; vortex. 5. Add 250 µl lysis solution; invert 6–8x. 6. Add 350 µl neutralization solution; invert 6–8x. 7.
Section 7 Troubleshooting Guide Problem Possible Cause Possible Solution Low plasmid yields Low copy number plasmid Use high copy number constructs Poor plasmid propagation in culture Inoculate large-scale cultures with overnight cultures generated from fresh colonies grown on a selective medium Determine optimum growth and plasmid propagation times for culture depending upon host, broth, etc.
Problem Possible Cause Possible Solution Bacterial DNA contamination Excessive amount of bacteria processed Determine OD600 of culture and do not exceed 12 OD•ml of bacteria processed Excessive agitation of lysate Do not shake or vortex lysate after addition of lysis solution Excessive amount of bacteria processed Determine OD600 of culture and do not exceed 12 OD•ml of bacteria processed Compromised RNase activity due to age or storage conditions Replace kit Excessive amount of bacteria process
Problem Possible Cause Possible Solution More than one band Presence of multimers – Try different host or on analytical gel typical and variable growth conditions depending upon plasmid, bacterial host, etc.
