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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. Product Specifications 4
Poly A (Carrier RNA)

X. Principle 4
XI. Materials and Equipment Needed But Not Provided 5
XII. Protocols
Before You Begin 6
DNA Extraction from Forensic Sample 6
DNA Extraction from Small Amount of Tissue 10
DNA Extraction from Urine 11
DNA Extraction from Small Volumes of Blood and Saliva 12
DNA Clean-Up 13
XIII. Troubleshooting guide 15
XIV. Ordering information 17
XV. Explanation symbols 18
I. Overview

Description
MagListoTM 5M Forensic Sample DNA Extraction Kit utilizes Magnetic Nano Beads to extract
total DNA from a variety of forensic sample, such as whole blood, saliva, dried body fluid
TM
spot, fingerprint, nail clipping or hair using Magnetic Nano Beads and MagListo Magnetic
TM
Separation Rack. The use of MagListo Magnetic Separation Rack along with this kit
greatly increases user’s convenience by saving process time without use of centrifuge.

Features and Benefits


- Magnetic Nano Beads enable rapid extraction
TM
- No expensive instrument required. Our MagListo Magnetic Separation Rack is very cost
conscious.

Applications
PCR, Real-Time PCR, SNP genotyping, STR (Short Tandem Repeat) analysis

II. Kit Components

MagListoTM 5M Forensic Sample DNA Extraction Kit K-3614 K-3615


Cat. no. 3614, 3615 (8 reactions) (100 reactions)

Buffer ① (Lysis) 4 ml x 1 ea 40 ml x 1 ea

Buffer ② (Binding) 3 ml x 1 ea 30 ml x 1 ea

st
Buffer ③ (1 Washing) 4 ml x 1 ea 40 ml x 1 ea

nd
Buffer ④ (2 Washing) 1.6 ml x 1 ea 16 ml x 1 ea

Buffer ⑤ (Elution) 1 ml x 1 ea 15 ml x 1 ea

Magnetic Nano Bead - DNA 1 ml x 1 ea 1.8 ml x 6 ea

Proteinase K 5 mg x 1 ea 25 mg x 2 ea

Poly A (Carrier RNA) - 1 mg x 1 ea

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III. Storage
MagListoTM 5M Forensic Sample DNA Extraction Kit should be stored dry at room
TM
temperature. It can be stored for up to 1 year, if it remains sealed. MagListo 5M Forensic
Sample DNA Extraction Kit provides optimized Buffer ② (Binding) which is poisonous and
hazardous. Please, wear gloves and goggle eye protection when working with Buffer ②
(Binding).

IV. Intended Use


MagListoTM 5M Forensic Sample 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 Material Safety Data
Sheet (MSDS) of this product.

Before, during and after use of this kit as described in this User’s Guide, all potentially
hazardous materials (i.e. materials that may have come in contact with genetically
recombinant samples) including tubes and tips should be processed and disposed of
according to applicable and appropriate regulations of the municipality/government in which
this product is being used. Users must be trained with basic experimental techniques for
correct execution of the experiments described in the User’s Guide.

Some applications that may be performed with this kit may infringe upon existing patents in
certain countries. The purchase of this kit does not include or provide a license to perform
patented applications. Users may be required to obtain a license depending on country and
application. We do not condone nor recommend unlicensed use of a patented application.

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 any issues are discovered relating to

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compromise in product quality, immediately contact BIONEER’s Customer Service Center
(order@bioneer.com).

BIONEER does not assume liability for misuse of the product, i.e. usage of the product for
any purposes other than its intended purpose as described in the User’s Guide. BIONEER
assumes liability under the condition that users disclose all information related to the
problem in written form within 30 days of occurrence.

VII. Technical Assistance


At Bioneer, we pride ourselves on being responsive to customers’ needs. If you have any
TM
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: sales@bioneer.com

In North America
Tel: +1-877-264-4300
Email:support@bioneer.us.com

VIII. Quality Management


Every aspect of our quality management system from product development, production to
quality assurance and supplier qualification meets the world-class standards. Each lot of
MagListoTM 5M Forensic Sample DNA Extraction Kit is carefully tested by our quality control
team.

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IX. Product Specifications

Feature Specification

Sample type Various kind of forensic sample

Technology Magnetic Nano Bead

Hands-on time < 10 min

Expected purity A260/280 > 1.8

Poly A (Carrier RNA)


MagListoTM 5M Forensic Sample DNA Extraction Kit is able to extract total DNA from very
4
small amounts of sample. If the sample contains low copy of cells (< 1x10 ) or has a small
amount of DNA (<100 ng), we recommend adding 2 μg of poly A (KB-3415-7) to the
sample after Buffer ② (Binding). Carrier RNA, such as poly A, enhances binding of DNA to
Magnetic Nano Beads.
To prepare poly A solution please add 1 ml of nuclease free water to the tube containing
lyophilized poly A (KB-3415-7) to obtain a solution of 1 μg/μl. Dissolve poly A completely,
divide it into conveniently sized aliquots and store at –20°C. Freeze-thaw repetition of the
aliquots more than 3 times will result in degradation of RNA. Poly A can be removed later
by RNase digestion. If the sample contains large amount of DNA, addition of poly A is not
necessary.

X. Principle
MagListoTM 5M Forensic Sample DNA Extraction Kit is designed for extraction of total DNA
from a variety of sources including high molecular weight up to 40 Kb (Note: this is
common for most DNA based application). Overall principle is based on adsorption of DNA
onto Magnetic Nano Beads by chaotrophic salt. For instance, chaotropic agent in Buffer ②
(Binding) contains guanidine hydrochloride. This removes water molecules around DNA and
silica coated Magnetic Nano Beads surface, resulting in total DNA being captured by
Magnetic Nano Beads. Magnetic Nano Beads and nucleic acid complexes are then pulled
and fixed on the tube wall using a magnetic force, followed by washing with ethanol to
remove debris and excessive salts. Captured nucleic acids are then eluted by Buffer ⑤

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(Elution), which contains an aqueous solution with optimal pH.

Sample Lysis Binding Washing Elution

XI. Materials and Equipment Needed But Not Provided


TM
1. 1 ml tube with 8-cap strip, 1.5 ml or 2 ml tubes based on sample size and MagListo
Magnetic Separation Rack
2. Vortex mixer
3. Absolute ethanol
4. Thermal block or dry oven
5. Phosphate-buffered saline (PBS)
6. Blow dryer or heat gun
7. MagListoTM Magnetic Separation Rack with appropriate size

Magnetic Separation Rack Choice

Tube MagListoTM Magnetic Separation Rack


MagListoTM-8Ch Magnetic Separation Rack
1 ml tube with 8-cap strip
(Cat. #. TM-1000)
MagListoTM-2 Magnetic Separation Rack
1.5 ml or 2 ml microcentrifuge tube
(Cat. #. TM-1010)

Please refer to the ordering information table on the latter part of the
manual, which contains the appropriate catalog number for specific tube
size.

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XII. Protocols

Before you begin


1. Completely dissolve Proteinase K in 250 ul (KB-3614-7) or 1,250 ul (KB-0111) of
nuclease-free water. Dissolved Proteinase K should be stored at 4℃.

2. Completely dissolve Poly A (KB-3615-7) in 1,000 ul of nuclease-free water. Divide it


into conveniently sized aliquots and store at –20°C.

3. Buffer ② (Binding) contains chaotropic salt. You should take the appropriate laboratory
safety precautions and wear gloves when handling.

st nd
4. Add correct amount of absolute ethanol to Buffer ③ (1 Washing) and ④ (2 Washing).

a. DNA Extraction from Forensic Sample

※ Handling Samples

Dried body fluid spot or fingerprint (FTA card, paper, cloth etc.)
Punch out the sample up to 7 mm diameter using single-hole paper puncher or cut out up
2
to 2 cm . Cut the sample into smaller pieces to increase lysis efficiency.
Hair
Cut the hair 1 cm length from the hair root. Without root, cut up to 5 strands of whole hair
into small pieces.
Bone & teeth
Homogenize the bone up to 100mg into fine powder.
Chewing gum
Cut up to 30 mg of chewing gum into small pieces.
Cigarette butts
2
Cut out up to 2 cm piece of outer paper from the end of the cigarette butt.
Buccal swab
Cut the swab from its stick by hand or scissors. Use single piece of swab for extraction.

1. Apply the forensic sample to a 1.5 ml or 2 ml tube.

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2. (Lysis: 2-7) Add 300 μl of Buffer ⓛ (Lysis) and 10 μl of proteinase K solution (see
“Before you begin”) to the tube and completely resuspend the contents with vortex
mixer or pipetting.

3. (Optional) Add 20 μl of 1M DTT (Dithiothreitol, Not provided) to the tube and completely
resuspend the contents with vortex mixer. If the sample is hair, nail clipping or semen
stains, this step is necessary to increase sensitivity.

4. Incubate at 60℃ for at least 1 hour.


(Note) Incubation time for complete lysis varies depending on the type of sample used
and age of starting material. If the sample is nail clipping, bone or considerably old,
extend incubation time up to overnight.

5. Add 300 μl of Buffer ② (Binding) to the tube and completely resuspend the contents by
vortex mixer or pipetting.

6. (Optional) Add 2 μl of dissolved poly A (see “Before you begin” or page 4 for details) to
the tube.

7. Incubate at 60℃ for 20 min.

8. Centrifuge at 13,000 rpm for 1 min to obtain clear lysate.

9. Take the clear supernatant only and transfer into new 1.5 ml or 2 ml tube.

10. (DNA Precipitation) Add 600 μl of absolute ethanol and mix well using a vortex mixer or
pipetting.

11. (DNA binding with Magnetic Nano Bead: 11-13) Add 100 μl of Magnetic Nano Bead
solution to the tube and mix thoroughly using a vortex mixer until Nano Beads are fully
resuspended.
(Note) Magnetic Nano Bead solution should be shaken well before each use.

TM
12. Place the tube in MagListo -2 Magnetic Separation Rack with magnet plate attached

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and invert the Rack with tube 3-4 times gently until Nano Beads bind tightly to magnet.
- Attachment

Combine magnet plate and the stand.

TM
13. Without removing the tube from MagListo rack, carefully pour supernatant out and
completely remove the remaining supernatant using a paper towel. In process,
magnetic crude pellet remains attached to the side of tube.
- Discard solution

Discard solution by inverting MagListo™ rack. Silicone immobilizer inside the stand holds
the tubes from falling in an upside down position. When discarding solution, invert rack
completely so the solution does not smear on the rack.

st TM
14. (1 washing: 14-16) Detach magnet plate from MagListo stand. Add 700 μl of Buffer
st
③ (1 Washing) to the tube. Close the cap and mix with vortex mixer until Nano Beads
are fully resuspended.
- Detachment

Push magnet plate upward gently.

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TM
15. Attach the magnet plate to MagListo stand and invert the Rack with tube 3-4 times
gently until Nano Beads bind tightly to magnet.

TM
16. Without removing the tube from MagListo rack, pour supernatant out and remove
remaining supernatant using a paper towel by blotting.

nd nd
17. (2 washing) Repeat the above step 14 - 16 by adding 700 μl of Buffer ④ (2 Washing)
for additional washing.

rd
18. (3 washing) Repeat the above step 14 - 16 by adding 700 μl of absolute ethanol for
additional washing.

19. (Drying) Completely dry Nano Beads with the tube open and use a heat gun or a blow
dryer for 1 min 3 cm away from the top of the tube.
(Note) If you do not have using a heat gun or a blow dryer, place the rack lying down in
a dry oven at 60℃ for 10 min.

20. (Elution: 20-24) Add 30 - 100 μl of Buffer ⑤ (Elution) or distilled water to the tube with
magnet plate detached and resuspend the contents completely by pipetting or vortex
mixer for 15 sec.

21. Incubate the tube at 60℃ for 1 min.

TM
22. Attach magnet plate to MagListo stand and invert the Rack with tube 3-4 times gently
until Nano Beads bind tightly to the magnet.

TM
23. Without removing the tube from MagListo rack, carefully transfer supernatant
containing DNA to a sterile microcentrifuge tube.

24. Discard used Magnetic Nano Beads. Do not reuse Nano Beads.

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b. DNA Extraction from Small Amount of Tissue

1. (Homogenization) Disrupt (or homogenize) the sample (~ 10 mg) with homogenization


tool. Place them into a 1.5 ml or 2 ml tube immediately.
(Note) Grind the tissue completely into fine powder with mortar and pestle or other
homogenization tool under liquid nitrogen. Final yield of DNA depends on the
amount and the type of the used tissue.

2. (Lysis: 2-6) Add 90 μl of Buffer ⓛ (Lysis).

3. Add 10 μl of proteinase K solution (see “Before you begin”) to the tube and mix
thoroughly using a vortex mixer.

4. (Optional) If RNA-free DNA is required, add up to 200 ug of RNase A (KB-0101, not


provided) and incubate for 2 min at room temperature.

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

6. Add 100 μl of Buffer ② (Binding) to the tube and mix immediately and thoroughly using
a vortex mixer. .

7. (DNA Precipitation) Add 200 μl of absolute ethanol and mix well using a vortex mixer or
pipetting.

8. Go to step 11 of “DNA Extraction from Forensic Sample” in page 7 and continue


extraction process.

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c. DNA Extraction from Urine

1. (Cell collection: 1-3) Centrifuge the urine and discard supernatant.


a. (~ 2ml) Centrifuge at 8,000 rpm (6,000 xg) for 2 min.
b. (~ 15ml) Centrifuge at 3,500 rpm (2,000 xg) for 10 min.

2. Add 500 ul of PBS and completely resuspend with vortex mixer or pipetting. Transfer the
sample to a 1.5 ml or 2 ml tube.

3. Centrifuge at 8000 rpm for 2 min and discard supernatant.

4. (Lysis: 4-8) Add 300 μl of Buffer ⓛ (Lysis) and 10 μl of proteinase K solution (see
“Before you begin”) to the tube and completely resuspend the contents with vortex
mixer or pipetting.

5. (Optional) Add 10 μl of 1M DTT to the tube and completely resuspend the contents with
vortex mixer. If the urine is expected to contain sperm cells, this step will improve yield.

6. Incubate at 60℃ for 1 hour.

7. Add 300 μl of Buffer ② (Binding) to the tube and completely resuspend by vortex mixer.

8. Incubate at 60℃ for 10 min.

9. (DNA Precipitation) Add 600 μl of absolute ethanol and mix well using a vortex mixer or
pipetting.

10. Go to step 11 of “DNA Extraction from Forensic Sample” in page 7 and continue
extraction process.

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d. DNA Extraction from Small Volumes of Blood and Saliva

1. Add 10 μl of proteinase K solution (see “Before you begin”) to 1ml, 1.5 ml or 2 ml tube.

2. Apply 1~100 μl of blood or saliva to the tube containing proteinase K.


(Note) If the sample volume is less than 100 ul, make the total volume 100 μl by adding
1X PBS to achieve maximum lysis efficiency and yield.

3. (Lysis: 3-4) Add 100 μl of Buffer ② (Binding) to each sample and mix immediately and
thoroughly using a vortex mixer. You must completely resuspend the sample to achieve
maximum lysis efficiency.

4. Incubate at 60℃ for 10 min.

5. (DNA Precipitation) Add 200 μl of absolute ethanol and mix well using a vortex mixer or
pipetting.

6. Go to step 11 of “DNA Extraction from Forensic Sample” in page 7 and continue


extraction process.

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e. DNA Clean-Up

1. Transfer the eluate or enzyme reaction product to 1.5 ml or 2 ml tube.

2. (Optional) If RNA-free DNA is required, add up to 200ug of RNase A (KB-0101, Not


provided) and incubate for 2 min at room temperature.

3. (Binding) Add 1 volume of Buffer ② (Binding) to the eluate and mix completely with
vortex mixer.

4. (DNA precipitation) Add 3 volumes of absolute ethanol to the eluate and mix well with
vortex mixer.

5. (DNA binding with Magnetic Nano Bead: 5-7) Add 100 μl of Magnetic Nano Bead
solution to the tube and mix thoroughly using a vortex mixer until Nano Beads are fully
resuspended.
(Note) Magnetic Nano Bead Solution contains magnetic nano beads. Please shake well
before use.

6. Place the tube in MagListo™-2 Magnetic Separation Rack with the magnet plate
attached and invert the Rack with tube 3~4 times gently until Nano Beads bind tightly to
magnet.

- Attachment

Combine magnet plate and the stand.

7. Without removing the tube from MagListo™ rack, carefully pour supernatant out and
completely remove remaining supernatant using paper towel by blotting.

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- Discard solution

Discard solution by inverting the MagListo™ rack. Silicone immobilizer inside the stand
holds the tubes from falling in an upside down position. When discarding solution,
invert rack completely so the solution does not smear on the rack.

st
8. (1 washing: 8-10) Detach the magnet plate from MagListo™ stand. Add 700 μl of
nd
Buffer ④ (2 Washing) to the tube. Close the cap and mix with vortex mixer until Nano
Beads are fully resuspended.
- Detachment

Push magnet plate upward gently.

9. Attach magnet plate to MagListo™ stand and invert the tube 3~4 times gently until
beads bind tightly to magnet.

10. Without removing the tube from MagListo™ rack, pour supernatant out and remove
remaining supernatant using paper towel by blotting.

11. Go to step 18 of “DNA Extraction from Forensic Sample” in page 9 and continue clean-
up process.

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XIII. Troubleshooting Guide

Comments and suggestions

Buffers or other reagents may have been exposed to external


changes or conditions that reduce its effectiveness. Please make sure
that reagents were stored at room temperature at all times upon
arrival and all reagent bottles were closed tightly, in order to preserve
pH and stability and to avoid contamination.

st
Please check and add ethanol to the Buffer ③ (1 Washing) and ④
nd
(2 Washing). After adding ethanol, mix Washing Buffer well and
always mark the Washing Buffer bottles. If not, Washing Buffer
remain concentrated and may wash away the adsorbed DNA.

The lysis may have been incomplete. Please extend the incubation
Low yield of DNA
time. The amount of time for complete lysis varies depending on the
type of sample used and age of starting material. A shaking water
bath should be used for efficient lysis.

Elution may have been incomplete. Please extend incubation time up


to 3 minutes at elution step to improve yield. In addition, make sure
that Magnetic Nano Beads are resuspended completely in the eluate
during incubation.

Pellet of Magnetic Nano Bead could be lost while discarding solution.


Check that all of the Nano Beads bind tightly to magnet when you
discard solution.

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Beads may have been dried insufficiently. You must completely dry
Nano Beads in a drying step. Remained ethanol can decrease purity
of DNA. Take enough time to completely dry Nano Beads.

Low A260/280 ratio

Incomplete suspension of beads during the washing step causes salts


to remain in purified DNA. Make sure Nano Beads are suspended
thoroughly during the washing process.

MagListo™ 5M Forensic Sample DNA Extraction Kit copurify DNA and


RNA when both are present in the sample. If RNA-free DNA is
required, 200ug of RNase A (KB-0101, not provided) should be
Copurification of RNA
added to each sample before addition of Buffer ② (Binding). If you
want to remove RNA in the eluate, refer to “DNA Clean-Up” protocol
in page 13 for details.

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


There is a white precipitate
after prolonged storage at low temperature. Incubation at 60℃ should
in some buffer
dissolve any precipitate in Buffer ⓛ or Buffer ②.

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XIV. Ordering Information

Cat no. Product Description Size

K-3601SM MagListoTM 5M Plamid Extraction Kit, 8 reactions (mini) 1 kit

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-3602 MagListoTM 5M Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit

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

K-3604 MagListoTM 5M Plant Genomic DNA Extraction Kit, 8 reactions (mini) 1 kit

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

K-3606 MagListoTM 5M Gel Extraction Kit, 8 reactions (mini) 1 kit

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

K-3608 MagListoTM 5M PCR Purification Kit, 8 reactions (mini) 1 kit

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

K-3610 MagListoTM 5M Cell Total RNA Extraction Kit, 8 reactions (mini) 1 kit

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

K-3614 MagListoTM 5M Forensic Sample DNA Extraction Kit, 8 reactions (mini) 1 kit

K-3615 MagListoTM 5M Forensic Sample DNA Extraction Kit, 100 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

15 ml tube x 6
TM-1020 MagListoTM-15 Magnetic Separation Rack
holes

50 ml tube x 3
TM-1030 MagListoTM-50 Magnetic Separation Rack
holes

TM-1040 MagListoTM-96 Magnetic Separation Rack 96-well plate 1ea

MagListoTM-2,
TM-1100 MagListoTM Magnetic Separation Rack Bundle Set -15, -50, and -96
(4 racks, 1 each)
K-3601-A Blow Dryer 1 ea

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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XV. Explanation Symbols

Contains
Catalog Consult Instruction
sufficient for (n) USE BY
Number For Use
tests

Caution, consult
Temperature
Batch code accompanying Research Use Only
Limitation
documents

Caution,
DO NOT
Manufacturer Potential
REUSE
Biohazard

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