Kit for the extraction of genomic DNA from blood and ... · BQ1 101 01 Revision : 2 (2016-11-04) I. Overview Description MagListo™ 5M Genomic DNA Extraction Kit utilizes Magnetic

Kit for the extraction of genomic DNA from blood and tissue using MagListo™

Version No.: 2.0 (2017-03)

Please read all the information in booklet before using the unit

Bioneer Corporation

8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon

34302, Republic of Korea

Tel: +82-42-930-8777

Fax: +82-42-930-8688

Email: [email protected]

www.bioneer.com

MagListo™ is a trademark of Bioneer Corporation.

Copyright 2017. Bioneer Corporation. All Rights Reserved.

mailto:[email protected]

Contents

I. Overview 1

II. Kit components 1

III. Storage 2

IV. Intended use 2

V. Safety warnings and precautions 2

VI. Warranty and liability 2

VII. Technical assistance 3

VIII. Quality management 3

IX. Kit specifications 4

Extraction of genomic DNA small amount of sample 4

Recommended amounts of starting sample 4

X. Sample preparation 5

XI. Principle 6

XII. Magnetic Nano Bead Information 6

XIII. Guidelines for MagListo™ Magnetic Separation Rack 7

XIV. Materials and Equipment Needed But Not Provided 8

Types of the Magnetic Separation Rack 8

XV. Procedure 9

XVI. Protocols 10

Before You Begin 10

A. DNA Extraction from Whole Blood 10

B. DNA Extraction from Cultured Cell 15

C. DNA Extraction from Animal Tissue 20

D. DNA Extraction from Bacterial cells (Gram-Negative Bacteria) 25

E. DNA Extraction from Bacterial cells (Gram-Positive Bacteria) 26

F. DNA Clean-Up (DNA Purification) 27

XVII. Appendix 29

Troubleshooting guide 29

Experimental data 32

XVIII. Ordering information 35

XIX. Explanation symbols 36

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I. Overview

Description

MagListo™ 5M Genomic DNA Extraction Kit utilizes Magnetic Nano Beads to extract total DNA from

various of sources, such as whole blood, animal tissue and cultured cells, with the aid of the MagListo™

Magnetic Separation Rack. The use of MagListo™ Magnetic Separation Rack along with the kit greatly

increases user convenience by shortening the extraction time without centrifugation.

Features and Benefits

- Magnetic Nano Beads enable the rapid nucleic acid extraction

- No requirement of expensive instruments

- A single kit serves mini or midi scale experiment

Applications

Gene Cloning, PCR, Real-Time PCR, Southern Blotting, SNP genotyping

II. Kit Components

MagListo™ 5M Genomic DNA Extraction Kit *K-3603

Buffer ① (Lysis) 25 ml x 1 ea

Buffer ② (Binding) 25 ml x 1 ea

Buffer ③ (1st Washing) 120 ml x 1 ea

Buffer ④ (2nd

Washing) 80 ml x 1 ea

Buffer ⑤ (3rd Washing) 120ml x 1 ea

Buffer ⑥ (Elution) 25 ml x 1 ea

Magnetic Nano Bead - DNA 1.8 ml x 6 ea

Proteinase K powder, lyophilized 25 mg x 2 ea

RNase A powder, lyophilized 24 mg x 2 ea

*Mini – 100 rxn, Midi – 15 rxn, Maxi – 8 rxn

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III. Storage

MagListo™ 5M Genomic DNA Extraction Kit should be stored dry at room temperature. It can be stored

for up to 2 years if it remains sealed.

IV. Intended Use

MagListo™ 5M Genomic DNA Extraction Kit is intended for research use only. This kit is not intended for

human or veterinary diagnostics.

V. Safety Warnings and Precautions

Please inquire BIONEER’s Customer Service Center to obtain a copy of the Material Safety Data Sheet

(MSDS) for this product.

Before, during and after using this kit, all potentially hazardous materials (i.e. materials that may have

come in contacted with genetically recombinant samples) including tubes, tips and other kit contents

should be processed and discarded in accordance with applicable and appropriate regulations of the

municipality/government in which this product is being used. A user must also be equipped with basic

experimental techniques required for correct execution of the extraction experiments described in this

User’s Guide.

Some inventions applied in this kit may infringe existing patents in certain countries. The purchase of this

kit does not include or provide a license to perform such patented inventions. Users may be required to

obtain a license depending on the patent law of country where this product is being used. We do not

condone nor recommend the unlicensed use of patented inventions.

VI. Warranty and Liability

All BIONEER products are manufactured and tested under strict quality control protocols. BIONEER

guarantees the quality of all directly manufactured products during the warranty period of one (1) year from

the date of purchase. If you find any issues regarding the product quality, please immediately contact

BIONEER’s Customer Service Center ([email protected]).

BIONEER does not assume liability for misuse of the product, i.e. using the product for any purposes

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other than its intended purpose as described in the User’s Guide. BIONEER will only assume liability under

the condition when the users disclose all related information regarding the issue to BIONEER in written

form within 30 days after occurrence of the issue.

VII. Technical Assistance

At Bioneer, we pride ourselves on being responsive to your needs. Our Technical Service Departments are

staffed by experienced scientists with extensive practical and theoretical expertise in Molecular Biology and

the use of Bioneer products. If you have any questions or would like to find out more information about

MagListo™ products, please contact us. We look forward to hearing from you!

Technical Support

For all technical questions and troubleshooting on Bioneer products and applications.

Tel: +82-42-930-8777

Email: [email protected]

In North America

Tel: +1-877-264-4300

Email:[email protected]

VIII. Quality Management

Every aspect of our quality management system from product development to supplier qualification

ensures that our products meet the world-class standards. Each lot of MagListo™ 5M Genomic DNA

Extraction Kit is carefully tested by the quality control team.

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IX. Kit Specifications

Sample Type Mini

(Typical yield)

Midi

(Typical yield)

Maxi

(Typical yield)

Whole blood 200 μl ( < 10 μg) 2 ml ( < 80 μg) 4 ml ( < 150 μg)

Cultured cell ~ 1 x 106 ( < 12 μg) ~ 5 x 10

6 (< 60 μg) ~ 1 x 10

7 (< 120 μg)

Animal tissue ~ 25 mg ( < 10 μg) ~ 100 mg ( < 40 μg) ~ 250 mg (< 120 μg)

Bacteria

(Gram (-), (+)) ~ 1 x 10

9 (< 15 μg) ~ 5 x 10

9 (< 80 μg) ~ 1 x 10

10 (< 150 μg)

Expected purity A260/280 > 1.8

* The DNA yield from samples with a low number of cells may be less than the figures shown in the table.

** For cultured cells, samples with cell number of ~104 can be used for “micro” scale extraction.

Extraction of genomic DNA from small amount of sample

MagListo™ 5M Genomic DNA Extraction Kit is able to extract genomic DNA from sample of low

quantity. If sample contains low number of cells (< 1x104) or has small amount of DNA, it is

recommended to add about 4 μg of carrier RNA to the starting sample. Ensure that the carrier DNA

does not interfere with your downstream application. Carrier RNA can be removed later by RNase

digestion. Please see “DNA Extraction from Cultured Cell for Micro” in page 15 for more details about

DNA extraction from samples with a low number of cells.

Recommended amounts of starting sample

It is recommended to use the amounts in Table 1 as starting sample amount.

Table1. Growth area and Average cell yield in various culture dishes.

Cell culture dishes Growth area (cm2) Average cell yield

Multi well plate

6 well 9.6 1.2 x 106

12 well 4 4 x 105

24 well 2 2 x 105

48 well 1 1 x 105

96 well 0.35-0.6 4 x 104

Dishes

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35 mm 8 1.2 x 106

60 mm 21 3 x 106

100 mm 55 8 x 106

150 mm 148 2 x 107

Flasks

50 ml 25 2.5 x 106

300 ml 75 1 x 107

X. Sample preparation

Several factors, such as harvesting method and storage of starting samples can influence the yield and

purity of DNA. All specimens must be stored in a freezer or used immediately after collection. It is

recommended to process the sample as soon as possible on ice, and to avoid repeated freezing and

thawing.

Blood

Blood sample should immediately be used or stored in a collection tube containing the anticoagulants

for blood (EDTA or ACDs). Samples can be stored for few days at 4℃ and at up to 1 year at -70℃. It is

recommended rapidly thaw frozen blood sample in water bath (37℃) and to keep the blood sample on ice

until use.

Cultured cells

Cultured cells can easily be harvested using a centrifuge. However, it might be difficult to extract

genomic DNA if cultured cells are too clustered. In this case, trypsin can be used to detach each cell from

the cluster. For extraction using MagListo™ 5M Genomic DNA Extraction Kit, number of cells should be

less than 1 x 107, which is calculated with a cell counter. It is recommended to keep samples on ice until

use.

Tissue

Tissue samples should immediately be used or stored at -70℃ upon harvest for optimal results. To

disrupt tissue sample, grind it in a pestle and a mortar with liquid nitrogen. Alternatively, a homogenizer or

a bead-beater can be used.

Bacterial cells

Bacterial cells can be processed in a shaking incubator for 12-16 hours at 37℃. Optimal results can be

obtained when harvested bacterial cells are immediately used or stored at between -20℃ and -80℃.

Additional bacteriolytic agents like lysozome or lysostaphin should be used to break the multilayered cell

wall of Gram-positive bacteria. For gram negative bacteria, these agents are not needed.

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XI. Principle

MagListo™ 5M Genomic DNA Extraction Kit is designed for the extraction of genomic DNA including high

molecular weight DNA (up to 40 kb). The overall principle is based on the adsorption of DNA onto the

Magnetic Nano Bead by chaotropic salt. For example, chaotropic agents in Buffer ② (Binding) contains

guanidine hydrochloride, as which removes water molecules around DNA and silica coated magnetic

beads surface resulting in genomic DNA then being captured by magnetic beads. The Magnetic Nano

Bead and nucleic acid complexes are pulled and fixed on the tube wall using a magnetic force, followed

by washing with ethanol to remove debris and excessive salts. Finally, the captured nucleic acids are then

eluted by Buffer ⑥ (Elution), an aqueous solution with an optimal pH.

Sample Lysis Binding Washing Elution

XII. Magnetic Nano Bead Information

Description

Magnetic Nano Beads have been developed to overcome shortcomings of existing resin and to automate

purification process. The principle of extraction using the Magnetic Nano Beads is that coated functional

group on the surface of the Magnetic Nano Beads bind DNA and With the Magnetic Nano Beads are then

isolated using external magnetic field.

Features

Fast binding guarantees higher throughput automation

Large surface area enables better sensitive assay

Globular structure increases specificity by decreasing non-specific binding

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Specification

AccuNanoBead™ Silica Magnetic Nano Beads

Matrix Silica-coated Fe3O4

Average size 400nm

Ligand -OH

Working Temp. 0-100℃

Storage Store at room temperature upon receipt

XIII. Guidelines for MagListoTM Magnetic Separation Rack

Description

MagListoTM Magnetic Separation Rack is designed for a fast, easy separation of the Magnetic

NanoBeads. Bioneer offers various racks of different sizes- MagListo™-8Ch for 8-tube strip and multi-

pipette, MagListo™-2 for 2 ml tube, MagListo™-15 for 15 ml tube, MagListo™-50 for 50 ml tube and

MagListo™-96 for 96 well microplate. These racks of different sizes allow users to choose the product

according to their needs.

The following are recommended when handling the MagListo™ Magnetic Separation Rack

The product is made of acryl and plastic. Be careful not to drop the product as the dropping may

break the product.

When moving the product, take extra care not to drop the product as it may cause injury.

If the product is breaks, do not discard it with bare hands as the sharp edges may cause injury.

When an extracted or purified nucleic acid is spilled on the product, immediately rinse it with running

water and clean it with 70% ethanol.

Acetone, Toluene, or organic solvent may damage the acrylic and plastic part of the product, which

may lead to malfunction of the product. Rinse the product immediately when spillage of any above

mentioned solvents occurs as the expected DNA yield may not be obtained if the product is

damaged.

Make sure that do not spill a corrosive liquid on the magnet plate part of the product. If spillage

occurs, immediately rinse it off with running water as it may corrode the magnet during storage and

may degrade its performance.

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XIV. Materials and Equipment Needed But Not Provided

1. 1.5 ml or 2 ml tube, 15 ml tube, 50 ml tube

2. Vortex mixer

3. Absolute ethanol

4. Thermal block or dry oven

5. Phosphate buffered saline (PBS)

6. MagListo™ Magnetic Separation Rack

Types of the Magnetic Separation Rack

Tube MagListo™ Magnetic Separation Rack

1 ml tube with 8-cap strip MagListo™-8Ch Magnetic Separation Rack

(Cat. no. TM-1000)

1.5 ml or 2 ml microcentrifuge tube MagListo™-2 Magnetic Separation Rack

(Cat. no. TM-1010)

15 ml tube MagListo™-15 Magnetic Separation Rack

(Cat. no. TM-1020)

50 ml tube MagListo™-50 Magnetic Separation Rack

(Cat. no. TM-1030)

(Note) Please refer to the ordering information in this User’s Guide for more information

regarding catalog number of racks designed for specific size of tubes.

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XV. Procedure-Genomic DNA Extraction

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XVI. Protocols

Before you begin

1. Completely dissolve Proteinase K (KB-0111) in 1,250 ul of nuclease-free water. Dissolved Proteinase

K should be stored at 4℃.

2. Completely dissolve RNase A (KB-3101) in 600 ul of nuclease-free water. Dissolved RNase A should

be stored at 4℃.

3. Buffer ② (Binding) and Buffer ③ (1st Washing) contain chaotropic salt. You should take appropriate

laboratory safety precautions and wear gloves when handling this buffer.

4. If there is any precipitate in Buffer ① (Lysis), incubate on a heating block at 56℃.

A. DNA Extraction from Whole Blood for Mini /Midi /Maxi scale

1. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see “Before you begin”) to each

tube.

a. (Mini) Add Proteinase K to a 1.5 ml or 2 ml tube.

b. (Midi) Add Proteinase K to a 15 ml tube.

c. (Maxi) Add Proteinase K to a 50 ml tube.

2. Transfer 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of whole blood and buffy coat to the tubes

containing proteinase K.

(Note) If the sample volume is less than the indicated volume above, bring the sample volume to a

total volume of 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) by adding 1X PBS to achieve maximum

lysis efficiency and yield.

3. (Lysis: 3-4) Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer ② (Binding) to each tube and

mix thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to

achieve maximum lysis efficiency.

4. Incubate the tubes at 60℃ for 10 min.

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5. (DNA Precipitation) Add 400 μl (mini) / 4 ml (midi) / 8 ml (maxi) of absolute ethanol to each tube

and mix well using a vortex mixer or by pipetting.

6. (DNA binding with Magnetic Nano Bead: 6-8) Add 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of

Magnetic Nano Bead solution to the each tube and mix thoroughly using a vortex mixer until the

beads are fully resuspended.

(Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix with a

vortex mixer before use.

7. Place the tube on the MagListo™-2 (mini) / MagListo™-15 (midi) / MagListo™-50 (maxi) Magnetic

Separation Rack with the magnet plate attached and invert the rack gently 3 to 4 times until the

beads bind tightly to the magnet.

- Attachment of the magnet plate

Combine the magnet plate to the stand.

8. Without removing the tubes from the MagListo™ Magnetic Separation Rack, discard the supernatant

carefully and completely remove the remaining supernatant on a paper towel by blotting action.

During this process, the magnetic crude pellet remains attached to the side of tube.

- How to discard the supernatant

Discard the supernatant by inverting the MagListo™ Magnetic Separation Rack. The silicone

immobilizer inside the stand prevents the tubes from falling. When discarding the supernatant, invert

the rack completely so that the solution does not to spill on the rack.

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9. (1st washing: 9-12) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add

500 μl (mini) / 3 ml (midi) / 5 ml (maxi) of Buffer ③ (1st Washing) to the each tube and close the

cap. Mix using a vortex mixer until the beads are fully resuspended.

- Detachment of the magnet plate

Detach the magnet plate gently by pulling it upwards.

10. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to the

magnet.


and remove the remaining supernatant on a paper towel by blotting action.

12. Repeat the steps 9 - 11 to remove remaining pigments and other debris.

13. (2nd

washing) Repeat the steps 9 - 11 by adding 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer

④ (2nd

Washing) instead of Buffer ③ for additional washing.

14. (3rd washing) Without removing the tubes from the MagListo™

Magnetic Separation Rack, add 700

μl (micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer ⑤ (3rd Washing) to “the opposite side of bead

pellet” close the cap and invert the rack twice in order to remove ethanol from the sample.

(Note) Direct pipetting of Buffer ⑤ onto the bead pellet, vortexing and/or vigorous shaking of the

tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected.

15. Discard the supernatant and completely remove the remaining supernatant by blotting action.

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- Add Buffer ⑤ and discard the supernatant

16. (Elution: 16-21) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 100

μl (mini) / 500 μl (midi) / 1 ml (maxi) of Buffer ⑥ (Elution) or distilled water to the each tube and

resuspend the bead pellet completely by pipetting or using a vortex mixer for 15 sec.


18. Vortex the tubes for 15 sec.

19. Place the tubes on the MagListo™ Magnetic Separation Rack, attach the magnet plate and invert

the rack gently 3 to 4 times until the beads bind tightly to the magnet.

20. Without removing the tube from the MagListo™ Magnetic Separation Rack, transfer supernatant

containing DNA carefully to a new sterile microcentrifuge tube.

21. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads.

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Summary of Reagents Volume in Each Step of DNA Extraction from Whole Blood

Step Buffer Mini Midi Maxi Page

Blood (+ PBS) 200 μl 2 ml 4 ml P. 10

Lysis Buffer ② (Binding) 200 μl 2 ml 4 ml P. 10

DNA Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 10

Bead Binding Magnetic Nano Bead - DNA 100 μl 500 μl 1 ml P. 11

1st Washing

(Repeat) Buffer ③ (1

st Washing) 500 μl 3 ml 6 ml P. 12

2nd

Washing Buffer ④ (2nd

Washing) 700 μl 5 ml 10 ml P. 12

3rd Washing Buffer ⑤ (3

rd Washing) 700 μl 8 ml 15 ml P. 12

Elution Buffer ⑥ (Elution) 100 μl 500 μl 1 ml P. 13

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B. DNA Extraction from Cultured Cells for Micro /Mini /Midi /Maxi scale

1. Centrifuge the cultured cells (~ 1x104 (micro) / ~ 1x10

6 (mini) / ~ 5x10

6 (midi) / ~ 1x10

7 (maxi)) for 10

min at 300 x g. Discard the supernatant carefully without disturbing the pellet.

(Note) For cultured cells with lower cell number than 1x104, “micro” scale as described in this

protocol.

2. Resuspend the pellet in 100 μl (micro) / 200 μl (mini) / 1 ml (midi, maxi) of 1X PBS and transfer the

resuspended cells to each tube.

a. (Micro / Mini) Transfer the resuspended cells to a new 1.5 ml or 2 ml tube.

b. (Midi / Maxi) Transfer the resuspended cells to a new 15 ml tube.

3. Add 10 μl (micro) / 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see “Before you begin”)

to the each tube.

4. If you require RNA-free genomic DNA is required, add up to 2 μl (micro) / 10 μl (mini) / 75 μl (midi) /

150 μl (maxi) of RNase A (see “Before you begin”) to each tube and incubate the tubes for 5 min at

room temperature.

5. (Lysis: 5-6) Add 100 μl (micro) / 200 μl (mini) / 1 ml (midi, maxi) of Buffer ② (Binding) to the each

tube and mix thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to

achieve maximum lysis efficiency.

6. Incubate the tubes at 60℃ for 10min.

7. (DNA precipitation) Add 200 μl (micro) / 400 μl (mini) / 2 ml (midi, maxi) of absolute ethanol to each

tube and mix well using a vortex mixer or by pipetting.

8. (DNA binding with Magnetic Nano Bead: 8-10) Add 100 μl (micro, mini) / 500 μl (midi) / 1 ml (maxi) of

Magnetic Nano Bead solution to each tube and mix thoroughly using a vortex mixer until the beads are

fully resuspended.

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(Note) Magnetic Nano Bead Solution contains Magnetic Nano Beads. Please shake well or mix with a


9. Place the tubes on the MagListo™-2 (micro, mini) / MagListo™-15 (midi, maxi) Magnetic Separation

Rack with the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly

to the magnet.








the rack completely so that the solution does not spill on the rack.


700 μl (micro, mini) / 5 ml (midi) / 10 ml (maxi) of Buffer ③ (1st Washing) to each tube and close the

cap. Mix using a vortex mixer until the beads are fully resuspended.

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magnet.



14. (2nd

washing) Repeat the steps 11 - 13 by adding 700 μl (micro, mini) / 5 ml (midi) / 10 ml (maxi)

Buffer ④ (2nd


15. (3rd washing) Without removing the tubes from the MagListo™

Magnetic Separation Rack, add 700 μl

(micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer ⑤ (3rd Washing) to “the opposite side of the bead

pellet”. Close the cap and gently invert the rack twice in order to remove ethanol from the sample.

(Note) Direct pipetting of Buffer ⑤ onto the bead pellet, vortexing and/or vigorous shaking of the

tubes may release nucleic acid from the beads, which may result in lower DNA yield than expected.

16. Discard the supernatant and completely remover the remaining supernatant by blotting action.


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17. (Elution: 17-21) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 50 μl

(micro) / 100 μl (mini) / 500 μl (midi) / 1 ml (maxi) of Buffer ⑥ (Elution) or distilled water to the each

tube completely resuspend the bead pellet by pipetting or using a vortex mixer for 15 sec.


19. Place the tubes on the MagListo™ Magnetic Separation Rack, attach the magnet plate and invert the

track gently 3 to 4 times until the beads bind tightly to the magnet.

20. Without removing the tubes from the MagListo™ Magnetic Separation Rack, transfer supernatant

containing DNA to a new sterile microcentrifuge tube.

21. Discard the tubes with remaining Magnetic Nano Bead pellet. Do not reuse the beads.

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Summary of Reagents Volume in Each Step of DNA Extraction from Cultured Cell

Step Buffer Micro Mini Midi Maxi Page

Cultured Cell ~ 1 x 104 ~ 1 x 10

6 ~ 5 x 10

6 ~1 x 10

7 P. 15

Lysis Buffer ② (Binding) 100 μl 200 μl 1 ml 1 ml P. 15

DNA Precipitation Absolute Ethanol 200 μl 400 μl 2 ml 2 ml P. 15

Bead Binding Magnetic Nano Bead -

DNA 100 μl 100 μl 500 μl 1 ml P. 15

1st Washing Buffer ③ (1

st Washing) 700 μl 700 μl 5 ml 10 ml P. 16

2nd


Washing) 700 μl 700 μl 5 ml 10 ml P. 17


rd Washing) 700 μl 700 μl 8 ml 15ml P. 17

Elution Buffer ⑥ (Elution) 50 μl 100 μl 500 μl 1 ml P. 18

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C. DNA Extraction from Animal Tissue for Mini /Midi /Maxi scale

1. (Homogenization) Disrupt (or homogenize) the tissue sample (~ 25 mg (mini) / ~ 100 mg (midi) / ~

250 mg (maxi)) with a mortar and a pestle. Transfer disrupted or homogenized sample to each tube.

a. (Mini) Transfer the homogenized tissue sample to a 1.5 ml or 2 ml tube.

b. (Midi) Transfer the homogenized tissue sample to a 15 ml tube.

c. (Maxi) Transfer the homogenized tissue sample to a 50 ml tube.

(Note) Immediately place the weighted, fresh or frozen tissue in liquid nitrogen and grind to a fine powder

with a mortar and a pestle. Final yield of DNA will depend on the amount and the type of the tissue

used.

2. (Lysis: 2-6) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Buffer ⓛ (Lysis) to each tube.

3. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see “Before you begin”) to the each

tube and mix thoroughly using a vortex mixer.

4. If you require RNA-free genomic DNA is required, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi)

of RNase A (see “Before you begin”) and incubate the tubes for 2 min at room temperature.

5. Incubate the tubes at 60℃ until the tissue is completely lysed.

6. Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer ② (Binding) to the each tube and mix

thoroughly using a vortex mixer. Make sure that you completely resuspend the sample to achieve

maximum lysis efficiency.

7. (DNA precipitation) Add 400 μl (mini) / 4 ml (midi) / 8 ml (maxi) of absolute ethanol to each tube and

mix well using a vortex mixer or by pipetting.


Magnetic Nano Bead solution to the each tube and mix thoroughly using a vortex mixer until the beads

are fully resuspended.

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(Note) Magnetic Nano Bead solution contains Magnetic Nano Beads. Please shake well or mix using a


9. Place the tubes on the MagListo™-2 (mini) / MagListo™-15 (midi) / MagListo™-50 (maxi) Magnetic

Separation Rack with the magnet plate and invert the rack gently 3 to 4 times until the beads bind

tightly to the magnet.




carefully and completely remove the remaining supernatant using a paper towel by blotting action.






700 μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer ③ (1st

Washing) to each tube and close the cap.

Mix using a vortex mixer until the beads are fully resuspended.

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magnet.



14. (2nd

washing) Repeat the steps 11 - 13 by adding 700 μl (mini) / 5 ml (midi) / 10 ml (maxi) Buffer ④

(2nd


15. (3rd washing) Without removing the tubes from MagListo™

Magnetic Separation Rack, add 700 μl

(micro, mini) / 8 ml (midi) / 15 ml (maxi) of Buffer ⑤ (3rd Washing) to “the opposite side of the bead

pellet”. Close the cap and invert the rack twice in order to remove ethanol from the sample.

(Note) Direct pipetting of Buffer ⑤ onto the bead pellet, vortexing or vigorous shaking of the tubes

may release nucleic acid from the beads, which may result in lower DNA yield than expected.

16. Discard the supernatant and completely remove the remaining supernatant by blotting action.


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17. (Elution: 17-21) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 100 μl

(mini) / 500 μl (midi) / 1 ml (maxi) of Buffer ⑥ (Elution) or distilled water to each tube and completely

resuspend the bead pellet by pipetting or using a vortex mixer for 15 sec.


19. Place the tubes on the MagListo™ Magnetic Separation Rack, attach the magnet plate and invert the

rack gently 3 to 4 times until the beads bind tightly to the magnet.

20. Without removing the tubes from MagListo™ Magnetic Separation Rack, transfer supernatant

containing DNA carefully to a new sterile microcentrifuge tube.

21. Discard the tubes with the remaining Magnetic Nano Bead pellet. Do not reuse the beads.

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Summary of Reagents Volume in Each Step of DNA Extraction from Animal Tissue

Step Buffer Mini Midi Maxi Page

Animal tissue ~ 25 mg ~ 100 mg ~ 250 mg P. 20

Lysis

Buffer ⓛ (Lysis) 180 μl 1.8 ml 3.6 ml P. 20

Buffer ② (Binding) 200 μl 2 ml 4 ml P. 20

DNA

Precipitation Absolute Ethanol 400 μl 4 ml 8 ml P. 20

Bead Binding Magnetic Nano Bead – DNA 100 μl 500 μl 1 ml P. 20

1st Washing Buffer ③ (1

st Washing) 700 μl 5 ml 10 ml P. 21

2nd


Washing) 700 μl 5 ml 10 ml P. 22


rd Washing) 700 μl 8 ml 15 ml P. 22

Elution Buffer ⑥ (Elution) 100 μl 500 μl 1 ml P. 23

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D. DNA Extraction from Bacterial Cells (Gram-Negative Bacteria) for Mini/Midi/Maxi

1. (Cell collection) Collect the bacterial cells (~1x109 (mini) / ~5x10

9 (midi) / ~1x10

10 (maxi)) by

centrifuging at 6000 x g (> 8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min).

Discard the supernatant (media) by using a pipette.

2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Buffer ⓛ (Lysis)

to the collected cell pellet and completely resuspend using a vortex mixer or by pipetting.

3. Transfer the cell suspension to each tube.

a. (Mini) Transfer the cell suspension to a 1.5 ml or 2 ml tube.

b. (Midi) Transfer the cell suspension to a 15 ml tube.

c. (Maxi) Transfer the cell suspension to a 50 ml tube.

4. Go to step 3 of “C. DNA Extraction from Animal Tissue” in page 20 and follow the instructions

accordingly.

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E. DNA Extraction from Bacterial Cells (Gram-Positive Bacteria) for Mini/Midi/Maxi

1. (Cell collection) Collect the bacterial cells (~ 1x109 (mini) / ~ 5x10

9 (midi) / ~ 1x10

10 (maxi)) by

centrifuging at 6,000 x g (>8,000 rpm in a microcentrifuge) for 10 min (or >3,500 rpm for 20 min).

Discard the supernatant (media) by using a pipette.

2. (Resuspension of cell pellet: 2-3) Add 180 μl (mini) / 1.8 ml (midi) / 3.6 ml (maxi) of Lysis Buffer for

Gram-Positive bacteria (not provided) to the collected cell pellet and completely resuspend using a

vortex mixer or by pipetting.

(Note) Lysis Buffer for Gram-Positive bacteria can be prepared by using this formulation: 20 mM Tris-

HCl (pH8.0), 2 mM sodium EDTA and 1.2% Triton® X-100

3. Transfer the cell suspension to each tube.

a. (Mini) Transfer the cell suspension to a 1.5 ml or 2 ml tube.

b. (Midi) Transfer the cell suspension to a 15 ml tube.

c. (Maxi) Transfer the cell suspension to a 50 ml tube.

4. (Lysis: 4-9) Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of lysozyme (100 mg/ml, not provided) to

each tube and mix thoroughly using a vortex mixer.

5. If you require RNA-free genomic DNA, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A

(see “Before you begin”).


7. Add 20 μl (mini) / 100 μl (midi) / 200 μl (maxi) of Proteinase K (see “Before you begin”) to each tube.

8. Add 200 μl (mini) / 2 ml (midi) / 4 ml (maxi) of Buffer ② (Binding) to each tube and mix thoroughly

using a vortex mixer.

9. Incubate the tubes at 60℃ for 30 min or until bacterial cells are completely lysed.


accordingly.

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F. DNA Clean-Up (DNA Purification)

1. Transfer the eluted DNA or enzyme reaction product to a new sterile tube.

a. (Mini) Transfer the eluate to a 1.5 ml or 2 ml tube

b. (Midi / Maxi) Transfer the eluate to a 15 ml tube

2. If you require RNA-free genomic DNA, add up to 10 μl (mini) / 75 μl (midi) / 150 μl (maxi) of RNase A

(see “Before you begin”) and incubate the tubes for 2 min at room temperature.

3. (Binding) Add 1 volume of Buffer ② (Binding) to 1 volume of the eluted DNA and mix completely


4. (DNA precipitation) Add 3 volumes of absolute ethanol to 1 volume of the eluted DNA and mix well



Magnetic Nano Bead solution to each tube and mix thoroughly using a vortex mixer until the beads are

fully resuspended.

(Note) Magnetic Nano Bead Solution contains Magnetic Nano Beads. Please shake well or mix with a


6. Place the tubes on the MagListo™-2 (mini) / MagListo™-15 (midi, maxi) Magnetic Separation Rack

with the magnet plate attached and invert the rack gently 3 to 4 times until the beads bind tightly to

magnet.



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8. (1st washing: 8-10) Detach the magnet plate from the MagListo™ Magnetic Separation Rack. Add 700

μl (mini) / 5 ml (midi) / 10 ml (maxi) of Buffer ④ (2nd

Washing) to each tube and close the cap. Mix

with a vortex mixer until the beads are fully resuspended.



9. Attach the magnet plate and invert the rack gently 3 to 4 times until the beads bind tightly to magnet.

10. Without removing the tubes from MagListo™ Magnetic Separation Rack, discard the supernatant and

remove the remaining supernatant on a paper towel by blotting action.


accordingly.

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XVII. Appendix

Troubleshooting guide

If you have problems during genomic DNA extraction, please use the Troubleshooting Guide. This

troubleshooting guide will help you to solve problem that may arise during DNA extraction. For other

technical assistance or more information, please contact our technical assistance team.

Comments and suggestions

Low yield of DNA

Buffers or other reagents may have been exposed to external factors that may

have reduced its quality. Please make sure that reagents are stored at room

temperature at all times upon arrival and that all reagent bottles are closed

tightly, in order to preserve pH and stability, and to avoid contamination.

Cells may not have been lysed properly, especially in the case of tissue cells.

Please, ensure that the turbid sample changes to a clear solution, indicating that

the protein digestion has occurred. Extend the incubation time if tissue cells are

still not lysed. The amount of time for complete lysis varies depending on the

type of tissue or sample type used. If a cell mass still remains after the overnight

incubation, centrifuge the sample and use supernatant for DNA extraction. A

shaking water bath should be used for efficient cell lysis.

Excess amount of starting sample was used to extract DNA. Appropriate amount

of starting sample (see “Kit Specification” in page 4) should be used for efficient

extraction of genomic DNA.

Elution may have been incomplete. Please extend incubation time up to 3

minutes at elution step to improve the DNA. In addition, make sure that Magnetic

Nano Beads are suspended completely in the eluting solution during incubation.

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Some of Magnetic Nano Bead pellet may have been lost while discarding

solution. Check that all of the Nano Beads have bound tightly to the magnet

when you discard supernatant.

Insufficient shaking or vortexing during lysis step may lead to low DNA yield.

Shake or mix with a vortex mixer sufficiently during incubation step.

Low A260/280 ratio

Beads may have been washed insufficiently. You must properly wash the beads

in the 3rd washing step. Remained ethanol can decrease the purity of DNA. Take

enough time to properly wash the beads.

Incomplete suspension of beads during the washing step causes salts to remain

in the purified DNA. Make sure that the beads are suspended thoroughly during

the washing process.

Presence of RNA in the

eluted DNA

RNA may be present in the eluted DNA when both DNA and RNA are resent in

the sample. If you require RNA-free genomic DNA, add RNase A should be

added to the sample before addition of Buffer ② (Binding). If you wish to RNA

from the eluted DNA, please refer to “F. DNA Clean-Up (DNA Purification)”

protocol in page 20 for details.

Excessively clustered

Magnetic Nano Bead

Excess amount of starting sample is used to extract DNA. Appropriate amount

of starting material (see “Kit Specification” in page 4) should be used for

efficient extraction of genomic DNA.

Presence of a white

precipitate in buffers

A white precipitate may form in Buffer ⓛ (Lysis) or Buffer ② (Binding) due to

prolonged storage at low temperatures. Incubate Buffer ⓛ (Lysis) or Buffer ②

(Binding) at 60℃ to dissolve any precipitate in the buffer.

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Degraded DNA

The DNA from old or incorrectly stored sample may often be degraded. As the

DNA yield is highly dependent on storage conditions of samples, please use

fresh samples for optimal results. In case of using stored tissue sample, it is

recommended to use sample stored at -70℃.

Frequent freezing and thawing may result in lower DNA yield than expected.

Avoid repeated freezing and thawing.

Flotation of extracted

DNA when loaded on

an agarose gel

Floating of DNA on an agarose gel is caused by the remaining ethanol in the

eluted DNA. Ensure that the 3rd Washing (ethanol removing) step in the protocol

is properly performed. Remaining ethanol may also interrupt the enzymatic

reaction.

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Experimental data

Figure1. Comparison of extracted genomic DNA from human whole blood (200μl) purified with MagListo™ 5M

Genomic DNA Extraction Kit and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6 7

M: Bioneer 1kb ladder

1-4: Extracted blood genomic DNA purified with Bioneer MagListo™ 5M Genomic

DNA Extraction Kit

5-7: Extracted blood genomic DNA purified with competitor Q kit

Figure2. Comparison of extracted genomic DNA from HeLa cell (1x106) purified with MagListo™ 5M Genomic

DNA Extraction Kit and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6


1-4: Extracted cell genomic DNA purified with Bioneer MagListo™ 5M Genomic DNA

Extraction Kit

5-6: Extracted cell genomic DNA purified with competitor Q kit

Sample Total Yield (ug) A260/A280 A260/A230

1 6.5 1.83 2.06

2 7.6 1.85 1.9

3 6 1.86 2.05

4 5.7 1.84 2.03

5 5.3 1.88 1.53

6 5.5 1.82 1.29

7 5.8 1.84 1.75


1 23.5 1.94 1.97

2 24.4 1.95 1.97

3 21.3 1.89 2.06

4 27.2 1.88 2.04

5 21.5 1.99 2.42

6 25.1 2 2.41

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Figure3. Comparison of extracted genomic DNA from bovine skeletal muscle tissue (15mg) purified with

MagListo™ 5M Genomic DNA Extraction Kit and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6


1-4: Extracted tissue genomic DNA purified with Bioneer MagListo™ 5M Genomic

DNA Extraction Kit

5-6: Extracted tissue genomic DNA purified with competitor Q kit

Figure4. Comparison of extracted genomic DNA from E. coli purified with MagListo™ 5M Genomic DNA

Extraction Kit and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6 7


1-2: (Mini) Extracted genomic DNA from E.coli (1x109) purified with Bioneer

MagListo™ 5M Genomic DNA Extraction Kit

3-4: (Mini) Extracted genomic DNA from E.coli (1x109) purified with

competitor Q kit 5-6: (Midi) Extracted genomic DNA from E.coli (5x10

9)

7: (Maxi) Extracted genomic DNA from E.coli (1x1010

)

Sample Total Yield (μg) A260/A280 A260/A230

1 5.7 1.84 1.53

2 5.2 1.86 1.7

3 5.6 1.87 1.76

4 5.4 1.88 1.76

5 5.5 1.87 1.69

6 5.7 1.82 1.08


1 14.4 1.84 1.49

2 15 1.82 1.39

3 15.6 2.05 1.45

4 15.3 2.04 1.55

5 94 1.76 1.51

6 73.8 1.84 1.37

7 141.6 1.78 1.51

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Figure5. Comparison of extracted genomic DNA from B. subtilis purified with MagListo™ 5M Genomic DNA

Extraction Kit and competitor’s kit (Single Column Type)

M 1 2 3 4 5 6


1-2: (Mini) Extracted genomic DNA from B. subtilis (1x109) purified with

Bioneer MagListo™ 5M Genomic DNA Extraction Kit

3-4: (Mini) Extracted genomic DNA from B. subtilis (1x109) purified with

competitor Q kit 5: (Midi) Extracted genomic DNA from B. subtilis (5x10

9)

6: (Maxi) Extracted genomic DNA from B. subtilis (1x1010

)

Sample Total Yield (μg) A260/A280 A260/A230

1 15.5 1.96 2.17

2 15 1.91 2.11

3 9.7 1.96 1.15

4 11.7 1.99 1.31

5 45.7 1.94 2

6 56.6 1.82 2.07

Figure6. Comparison of Real Time qPCR data of extracted genomic DNA from HeLa cell (1x101~1x10

5) purified

with MagListo™ 5M Genomic DNA Extraction Kit and competitor’s kit (Single Column Type)

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XVIII. Ordering Information

Cat no. Product Description Size

K-3601 MagListoTM 5M Plamid Extraction Kit, 100 reactions (mini) 1 kit

K-3600 MagListoTM 5M Plamid Extraction Kit, 500 reactions (mini) 1 kit

K-3603 MagListoTM 5M Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3605 MagListoTM 5M Plant Genomic DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3607 MagListoTM 5M Gel Extraction Kit, 100 reactions (mini) 1 kit

K-3609 MagListoTM 5M PCR Purification Kit, 100 reactions (mini) 1 kit

K-3611 MagListoTM 5M Cell Total RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3613 MagListoTM 5M Tissue Total RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3615 MagListoTM 5M Forensic Sample DNA Extraction Kit, 100 reactions (mini) 1 kit

K-3617 MagListoTM 5M Viral DNA / RNA Extraction Kit, 100 reactions (mini) 1 kit

K-3619 MagListoTM 5M Circulating Cell Free DNA Extraction Kit, 50 reactions (mini) 1 kit

TM-1000 MagListoTM-8Ch Magnetic Separation Rack 1 ml tube x 8 holes

TM-1010 MagListoTM-2 Magnetic Separation Rack 2 ml tube x 8 holes



HT-15-NG 1.5 ml microcentrifuge tube 500 ea / pk

HT-20-NG 2 ml microcentrifuge tube 500 ea / pk

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XIX. Explanation Symbols

Catalog

Number

Contains sufficient for

(n) tests USE BY

Batch code

Caution, consult

accompanying

documents

Temperature

Limitation

Manufacturer

Caution, Potential

Biohazard

DO NOT

REUSE

Consult Instruction

For Use

Documents

Kit for the extraction of genomic DNA from blood and ... · BQ1 101 01 Revision : 2 (2016-11-04) I. Overview Description MagListo™ 5M Genomic DNA Extraction Kit utilizes Magnetic