www.toyobo.co.jp/e/bio

F0983K

Instruction manual MagExtractor-Plasmid-2004

MagExtractor -PlasmidNPK-301

500 preparations

Store at 4°C

Contents[1]

Introduction[2]

Components[3]

Materials required[4]

Protocol1. Preparation of reagents required
2. Purification[5]

Troubleshooting[6]

References[7]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory precaution and utilize safety while using this kit.

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Tel(81)-6-6348-3888
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CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

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www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
MagExtractor -Plasmid- provides a simple and reliable method for the rapid purificationof plasmid DNA from E. coli cells utilizing magnetic silica beads. This kit is based on
binding properties of DNA to a silica surface in the presence of chaotropic agents1). The
purified plasmid can be used directly for automated fluorescent sequencing.

Fig. 1 Principle of purification

Features-Typical plasmid yield from an E. coli cell line carrying a high-copy plasmid is approx.
3-6 µg.
-This kit is suitable for high throughput extraction of plasmid from E. coli cells. The
extraction time is 10-15 minutes.
-Purified plasmid can be applied directly to sequencing, enzyme reaction, transformation,etc.
-This kit does not contain hazardous substances, such as phenol or chloroform.

Fig. 2 Flow chart of purification

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio[ 2 ] Components

This kit contains the following components for 500 preparations.
Binding Solution
Magnetic Beads I
Magnetic Beads II
Suspension Solution
Lysis Solution I
Lysis Solution II
Neutralization Solution
Elution Solution
5x Loading Dye

130 mL x 2 (store at 4°C or room temperature)
16 mL(store at 4°C or room temperature)
16 mL(store at 4°C or room temperature)
80 mL(store at 4°C）
80 mL
(store at 4°C or room temperature)
20 mL(store at 4°C）
65 mL
(store at 4°C or room temperature)
60 mL(store at 4°C)
11 mL
(store at 4°C）

Caution-“Binding Solution” contains chaotropic salt, which is an irritant. Lysis solution II
contains NaOH. Take appropriate laboratory safety measures and wear gloves whenhandling.
-“Suspension Solution” contains RNase A.
Notes-Magnetic Beads I are not necessary for manual use.
[This reagent is used for extraction with the automated nucleic acid purification
apparatus “MFX series”]-Lysis solution I and II should be mixed at a ratio of 4:1 prior to use. This solution canbe stored at room temperature for 3 weeks.
-If precipitates are formed in “Lysis Solution I” at low temperature, dissolve theprecipitates by heating at 40°C.
-Purified plasmid may contain small amounts of EtOH. Plasmid DNA solution
containing EtOH sink easily into agarose gel slots using 5x Loading Dye.

[3]

Materials required

The following materials are required for purification.
(1) Reagents-70% Ethanol(2) Instruments-Vortex mixer-Magnetic stand-(Heating block)Fig.3 Magnetic standMagical Trapper (Code No.MGS-101)

Notes-For complete evaporation of ethanol, a heating block at 78°C is necessary.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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www.toyobo.co.jp/e/bio[4]Protocol

1. Preparation of reagents required
Lysis solution I and II should be mixed at a ratio of 4:1 prior to use. This solution canbe stored at room temperature for 3 weeks.

2. Purification(1) Pellet 1.5-3 mL bacterial culture by centrifugation; discard as much supernatant
as possible.
Notes
Up to 3 mL of bacterial cultures can be used for high and low copy numberplasmids. Bacterial cultures should be incubated for 12-16 hours in appropriate
medium containing selection antibiotics.
(2) Resuspend pelleted bacterial cells thoroughly in 150 μL Suspension Solution.
(3) Add 150 μL Lysis Solution (mixed), and mix by inverting the tube 5 times (DONOT VORTEX)(4) Incubate on ice for 5 minutes.
(5) Add 120 μL l Neutralization Solution, and mix by inverting the tube 5 times (DONOT VORTEX)(6) Centrifuge at 12,000 rpm for 5 minutes(7) Carefully transfer all lysate to a fresh 1.5-mL microtube.
(8) Add 500 μL Binding Solution.
(9) <Binding> Add 30 μL Magnetic Beads II and vortex the tube for 1 minute.
Notes
Suspend magnetic beads completely prior to use.
(10) Place the tube in the magnetic stand. The magnetwill attract the magnetic beads, separating from thespecimen solution.
(11) After magnetic capture, carefully remove thesupernatant.

Fig. 4Magnetic separation(12) <Washing> Add 720 μL 70% EtOH to the tube and vortex for 10 seconds.
(13) Place the tube in the magnetic stand and collect the beads with the magnet.
(14) After magnetic capture, carefully remove the supernatant.
(15) <Washing> Repeat (12) - (14)(16) optional <Drying> Evaporate EtOH by heating the opened microtube to 78°C for≤ 15 minutes.

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www.toyobo.co.jp/e/bio(17) <Elution> Add 50 μL Elution Solution and vortex for 1 minute.
(18) Collect the supernatant and place in a fresh tube.
Notes:-Purified plasmid solutions that have not been heated contain small amounts of EtOH.
Plasmid DNA solution containing EtOH easily sink into agarose gel slots by using 5xLoading Dye.

[ 5 ] Troubleshooting

Symptom
Low yieldCauseSolution

Insufficient lysis of E.
coli cells
Low-copy plasmids

Insufficient lysis of E. coli cells decreases plasmidyields.
Increase the number of E. coli cells for purification.
When using low-copy plasmids, yields will be low.
Use DNase-gene deficient E. coli strains (e.g.,JM109, DH5α, and XL1-Blue). Plasmids from E.
coli strain carrying DNase-gene (e.g., HB101) might
be degraded during incubation.
Magnetic Beads I are not necessary for manual use.
This reagent is used in extraction with the automatednucleic acid purification apparatus “MFX series”.
Plasmid solutions purified without a drying step ight
contain small amounts of EtOH. Plasmid DNA
solutions containing EtOH easily sink into agarose
gel slots by using 5x Loading Dye.

Degradation of purified plasmid

Residual DNase

Unnecessary magnetic beads

Magnetic Beads I

Diffusion of DNA solution inelectrophoresis buffer

Trace amounts of EtOH
in the sample[ 6 ] References

1)

B. Vogelstein and D. Gillespie, Proc. Natl. Acad. Sci. USA. 76: 615-619 (1979)[ 7 ] Related products
Product namePackage
1 piece

Magnetic standCode No.
MGS-101

Magical Trapper

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

4