Quantum Prep
™
Plasmid Midiprep Kit
Instruction Manual
Catalog Number
732-6120
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547
4100111 Rev E
Table of Contents
Section 1 Introduction ..........................................1
1.1 Overview.........................................................1
1.2 Contents ..........................................................2
1.3 Storage and Stability.......................................2
Section 2 Protocol..................................................2
2.1 Recommendations for Best Results................3
2.2 Protocol...........................................................4
Section 3 References..............................................7
Section 4 Product Information.............................7
Section 1
Introduction
1.1 Overview
The Quantum Prep plasmid midiprep kit has been
optimized for the rapid purification of DNA from 40 to
50 ml of liquid culture. This kit uses the optimized
alkaline lysis protocol and patented DNA binding matrix
found in the Quantum Prep plasmid miniprep Kit.
matrix, made up of the silicon dioxide exoskeleton of
diatoms, results in DNA of the highest purity and yield
with the minimum of processing steps. A single midiprep
can be processed in approximately 45 minutes and requires
no ethanol precipitation. The Quantum Prep kit was
optimized using high-copy number plasmids grown in
either Luria Broth or Terrific Broth.
of culture and a single harvesting step. Multiple samples
can be processed simultaneously with minimal increase
in time. DNA purified using the Quantum Prep midiprep
kit can be used directly for enzymatic modification, cell
transfection and transformation, fluorescent sequencing
and in vitro transcription. Each lot is qualified for
automated fluorescent sequencing, yield, and enzymatic
manipulation. Yields of high copy plasmid are in the range
of 100 µg to over 300 µg from 40 ml of culture.
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It requires only 40 ml
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This
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1.2 Contents
The Quantum Prep plasmid midiprep kit contains sufficient reagents for 20 plasmid midipreps. Additional
Midiprep Wash Buffer will be required if following recommendations for EndA+ strains such as HB101 and
MC1061.
Cell Resuspension Solution 110 ml
Cell Lysis Solution 110 ml
Neutralization Solution 110 ml
Quantum Prep matrix 20 ml
Midiprep Wash Buf fer 125 ml
Spin columns and caps 20
2 ml microcentrifuge tubes 40
1.3 Storage and Stability
All components are guaranteed for 12 months from the
date of purchase, when stored at room temperature and
used as described in this manual.
Section 2
Protocol
Yields of plasmid DNA depend on factors such as
vector copy number, insert DNA, growth conditions, and
media. This midiprep protocol has been optimized for 40 ml
of culture of high-copy number plasmids grown in Luria
Broth or Terrific Broth. It is not recommended that larger
volumes of culture be used for medium to high-copy
plasmids, as that will not usually result in higher yields using
this protocol. For low-copy number plasmids (e.g. pBR322),
up to 60 ml of culture can be used with the volumes in this
protocol. Larger volumes may not result in higher yields
due to the increase of other cellular contaminants.
It is highly recommended that the plasmid DNA be
isolated from a host strain that contains a mutation in the
endonuclease I gene (endA) of E. coli. Isolation of DNA
from strains containing active endonuclease I gene product
(such as HB101 and MC1061) may result in samples which
contain trace amounts of nuclease. Additional precautions
and wash steps are required with these strains (see Section
2.1 and 2.2). For the highest quality DNA, these host strains
should be avoided.
2.1 Recommendations for Best Results
A precipitate may form in the lysis and neutralization
solutions due to cold temperatures from ambient winter
shipping conditions, cool laboratory temperatures, or factory
storage conditions. This will not affect the performance
of the product. If a precipitate is observed, warm the bottles
to 37 °C until it is redissolved. Use and store at room temperature.
• Add 125 ml of 95% ethanol to the Midiprep Wash
Buffer before first use.
• For increased plasmid yield a second elution may
be performed. Eluting with water or TE heated to
70 °C may also result in an improved yield.
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• To increase DNA concentration, elute with less
than the recommended elution volume or concentrate
the DNA by ethanol precipitation.
• If using EndA+ strains, grow in Luria Broth and
purify DNA from only 20 ml of culture using recommended volumes and extra wash steps noted.
2.2 Protocol
Note: All steps are performed at room temperature unless
otherwise indicated.
1. Inoculate plasmid-containing bacteria into 50 ml of
liquid media in a 250 ml Erlenmeyer flask. Incubate
overnight (15–18 hr) at 37 °C in a rotary shaker at
300 rpm.
2. Transfer 40 ml of the overnight culture to a 50 ml screwcap centrifuge tube. Spin down cells by centrifugation
for 5 minutes at 5,000 rpm (approximately 3,000 x g).
Discard the supernatant.
3. Add 5 ml of Cell Resuspension Solution to the cell
pellet. Vortex the cells to resuspend the pellet. Be
sure that the cell pellet is completely resuspended.
4. Add 5 ml of Cell Lysis Solution. Mix by inverting
the tube 6–8 times. Do not vortex, since this may
cause shearing of the chromosomal DNA, resulting
in contamination of the plasmid DNA. The solution
should become viscous and somewhat clear .
5. Add 5 ml of Neutralization Solution. Cap the tube and mix
by inverting the tube 6–8 times. The solution should
become cloudy and develop a flocculant white precipitate.
6. Centrifuge for 10 minutes at 8,000 rpm (7,500 x g).
Carefully pour the supernatant into a new tube. Try not
to transfer any of the precipitate. However, a small
amount of the debris will not affect plasmid purification.
7. Resuspend the Quantum Prep matrix by shaking
vigorously. Add 1.0 ml of Quantum Prep matrix to
the cleared lysate from step 6. Swirl gently for
15–30 seconds to mix. Centrifuge for 2 minutes at
8,000 rpm to pellet the matrix.
8. Carefully pour off the supernatant from the pelleted
matrix.
Add 125 ml of 95% ethanol to the wash buffer
before first use.
9. Add 10 ml of wash buffer to the matrix. Resuspend the
matrix in the wash buffer by shaking.
10. Centrifuge for 2 minutes at 8,000 rpm. Carefully pour
the wash buffer from the pelleted matrix. Add 600
µ l of wash buffer to the matrix and resuspend.
If isolating DNA from an EndA+ strain do an
additional 10 ml wash step at this point.
Additional wash buffer can be ordered using Bio-Rad
catalog number 732-6134. Alternatively the Midiprep
Wash Buffer, composed of 200 mM NaCl, 40 mM
Tris, 4 mM EDTA, pH 7.5 plus an equivalent amount
of 95% ethanol, may be made.
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11. Remove a spin column from the bag and snap off the
tab at the tip. Place the spin column inside a 2 ml collection tube. Transfer the resuspended matrix from
the previous step to the spin column. Puncture a
hole in a spin column cap with a small, sharp object
and cap the column with it. Spin 30 seconds in a
microcentrifuge at 12–14,000 x g. Remove the spin
column from its microcentrifuge tube, discard the
wash buffer at the bottom of the tube and replace
the filter in the same tube.
12. Add 500 µl of wash buffer and spin 30 seconds in a
microcentrifuge at 12–14,000g. Remove spin column
and discard Wash Buffer. If isolating DNA from an
EndA+ strain do two additional 500 µl wash steps.
13. Replace the spin column in the tube and spin an
additional 2 minutes at maximum speed to remove
any residual wash buffer.
14. Transfer the spin column to a clean 2 ml microcentrifuge tube. Add 600 µl of water or TE to the matrix.
Spin for 2 minutes in a microcentrifuge and discard the
spin column containing the matrix. Store plasmid
DNA at -20 °C.
Section 3
References
1. U.S. Patent 5,075,430 issued to Bio-Rad Laboratories.
2. Ausubel et al., Current Protocols in Molecular Biology,
Wiley-Interscience, New York (1987).
Section 4
Product Information
Catalog
Number Product Description
732-6137 Quantum Prep MaxiPrep Spin Baskets, 10
732-6134 Quantum Prep Maxi/Midiprep Wash Buffer,
270 ml
732-6120 Quantum Prep Plasmid Midiprep Kit, 20 preps
732-6122 Quantum Prep Neutralization Solution, 110 ml
732-6116 Quantum Prep Midi Spin Columns, 20 with caps
732-6024 Quantum Prep Wash Buffer, 250 ml
732-6100 Quantum Prep Plasmid Miniprep Kit, 100 preps
732-6110 Quantum Prep Matrix, 20 ml
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