Quantum Prep® Plasmid Miniprep Kit Instruction Manual Catalog #732-6100 For Technical Service Call Your Local Bio-Rad Office or in the U.S.
Table of Contents Section 1 Introduction.......................................................... 1 1.1 1.2 1.3 Overview.......................................................................... 1 Kit Contents..................................................................... 2 Storage and Stability........................................................ 3 2.1 2.2 2.3 Recommendations for Best Results................................. 3 Protocol.........................................................
Section 1 Introduction 1.1 Overview The original alkaline lysis method for purifying plasmid DNA from bacterial cultures requires organic reagents and timeconsuming steps to obtain high-quality DNA. The Quantum Prep plasmid miniprep kit has been optimized for the rapid purification of high-quality, high-yield plasmid DNA from 1–2 ml liquid cultures. This kit uses the silicon dioxide exoskeleton of diatoms as the DNA binding matrix.
Plasmid yield depends on a number of factors such as the vector copy number, culture volume and aeration, bacterial strain, and growth media. The Quantum Prep kit was optimized using high copy number plasmids grown in rich media such as Terrific Broth.2 The Quantum Prep kit permits yields as high as 25 µg from a 1.5 ml culture of DH5αF'[pTZ18U] grown in Terrific Broth. Yields as high as 40 µg have been obtained with other plasmids using the Quantum Prep kit. 1.
1.3 Storage and Stability All components are stable for 12 months from the date of purchase when stored at room temperature and used as described in this manual. Section 2 Protocol 2.1 Recommendations for Best Results • • • The lysis and neutralization solutions may exhibit salt precipitation due to cold temperatures. The product will not perform optimally if the salt precipitates out of the solution.
• Tris-HCl, 0.1 mM EDTA, pH 8.0). Eluting with H2O or TE preheated to 70°C can also improve the yield. However, these high temperatures may partially denature large plasmids. To increase the concentration of DNA eluted, use one half the recommended volume of H2O or TE (50 µl). Alternatively, the purified DNA may be ethanol-precipitated and resuspended in 10–20 µl of water or TE to achieve a higher concentration. See Figure 1 for a detailed comparison of DNA yield and concentration versus elution volume.
4. 5. 6. 7. should become viscous and slightly clear if cell lysis has occurred. Add 250 µl of the neutralization solution and mix by gently inverting the capped tube about ten times (do not vortex). A visible precipitate should form. Pellet the cell debris for 5 mins in a microcentrifuge. A compact white debris pellet will form along the side or at the bottom of the tube. The supernatant (cleared lysate) at this step contains the plasmid DNA.
The final formulation of the wash buffer contains 50% ethanol. To achieve this, add 63 ml of ethanol to the wash buffer before first use of the Quantum Prep kit. 8. Remove the spin filter from the 2 ml tube, discard the filtrate at the bottom of the tube, and replace the spin filter in the same tube. Add 500 µl of wash buffer and wash the matrix by centrifugation for 30 seconds. 9.
2.3 Helpful Hints 1. It is recommended that the cells do not sit longer than five minutes at step 3 (lysis) before proceeding to step 4. Additionally, it is recommended that the cells do not sit longer than ten minutes at step 4 (neutralization) before proceeding to step 5. 2. Due to the fixed angle for most microcentrifuges, the matrix may pellet against the side of the spin filter.
DNA concentration 400 25 350 20 300 250 15 200 10 150 100 DNA yield, µg DNA concentration, ng/µl 30 DNA yield 450 5 50 200 175 150 125 100 75 0 50 25 0 Elution volume, µl Fig. 1. Effect of elution volume on DNA concentration and yield from a 1.5 ml culture of DH5αF'[pTZ18U] grown in Terrific Broth and purified using the Quantum Prep Plasmid Miniprep kit.
Section 3 References 1. U.S. Patent 5,075,430 issued to Bio-Rad Laboratories. 2. Ausubel et al, Current Protocols in Molecular Biology, WileyInterscience, New York (1987).
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