LaboPassTMGelProtocol Book

LaboPass TM GELIntroduction
LaboPass TM Gel kit is designed for a fast and efficient isolation of 100 bp to10 kb of DNA fragments from TAE or TBE agarose gel. This kit can extract
up to 10 µg of pure DNA fragments from standard agarose gel as well as low
melting agarose gel. DNA fragments isolated with this kit can be directly
applied in ligation, labeling, or most of enzymatic reactions without further
manipulation.

Principle of methods
In the presence of high salt, the product DNA binds selectively to a glass
fiber membrane in a LaboPass TM Spin Column. The binding reaction occurs
due to the disruption of the organized structure of water molecules and theinteraction with the nucleic acid. The DNA remains bound while a series ofrapid "wash-and-spin" steps removes contaminating small molecules(primers, nucleotides and salts). Finally, a low salt buffer elutes the DNA
from the glass fiber membrane. The process does not require alcohol
precipitation, organic solvent extraction, or any further handling.

Storage and Stability

LaboPass TM Gel kit is shipped at room temperature. All solutions should be
stored at 15 – 25℃. The kit components are guaranteed to be stable for 12months from the date of manufacture printed on the product package.
Please note that improper storage at 4℃ (refrigerator) or -20℃ (freezer) will
adversely impact DNA purification when precipitates form in the buffers. The
Buffer GB may exhibit salt precipitation due to cold temperatures as shippingor lab ambient conditions in winter. The product will not perform optimally if
the salt precipitates out of solution. If that happen in any of the solutions,warm the bottles at 50℃ with occasional mixing until completely redissolved.

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Chemical Hazard
Buffer GB contains irritant which is harmful when in contact with skin or eyes,
or when inhaled or swallowed. Care should be taken during handling.
Always wear gloves and eye protector, and follow standard safety
precautions.

Quality Control
DNA molecular weight marker is separated on a 1 % agarose gel andfragments of various size are isolated according to the kit protocol. The
recovery of the isolated DNA fragments was at least 80 %. Isolated
fragments are readily digested with the restriction enzyme showing expected
pattern. All components in this kit are manufactured in particle-controlledarea (class 1000 clean room), and its degree of cleanness is monitored
periodically.

Kit Contents
Cat. No.
No. of preparation
Spin column
Collection tube
Buffer GB
Buffer NW(conc.)
Buffer EB

CMG0111
50
50
50
30ml
15ml
15ml

CMG0112
200
200
200
125ml
57ml
15ml

CMG0115
1000
1000
1000
125mlx557mlx515mlx5* All Solution (Buffer) is available to purchase separately.

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LaboPass TM GEL kit Protocols
Before experiment- All centrifugation should be carried out at 10,000 xg above (>12,000 rpm) at room

temperature in a microcentrifuge.
- Add absolute ethanol to Buffer NW as printed on the label before first use.
- All solutions should be equilibrated at room temperature before procedures.

1. Excise the DNA band of interest using an ethanol-cleaned razor
blade or scalpel on a transilluminator.
Minimize gel volume by cutting the gel slice as small as possible.

2. Weigh the gel slice in a microcentrifuge tube. Add 3 volumes ofBuffer GB to 1 volume of gel (100mg).
For example, add 300 µl of Buffer GB to each 100 mg of agarose gel slice.
For >1.5 % agarose gel, add 5 volumes of Buffer GB. If precipitated material
has formed in buffer GB, heat to dissolve at 37℃.

3. Incubate at 50℃ until the agarose gel is completely melted (510 minute). To help dissolve gel, mix by vortexing the tube every
2-3 minutes during the incubation.
4. After the slice has dissolved completely, check that the color of
the mixture is yellow. (similar to Buffer GB)
If the color of the mixture is dark orange or purple, add 10 µl of 3 M sodium
acetate, pH 5.0, and mix. The color of the mixture will turn yellow.

5. (Optional) Add 1 gel volume of isopropanol to the sample andmix. Transfer the mixture to a Spin Column.
This step increases the yield of DNA fragments < 500 bp and >4 kb. For
DNA fragments between 500 bp and 4 kb, addition of isopropanol has noeffect on yield.

6. Centrifuge for 1 min. Remove the Spin Column from the tubeand discard the flow through from the Collection Tube. Reinsertthe Spin Column into the Collection Tube.

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7. Add 750µl of Buffer NW to the Spin Column. Let it stand for 5minutes, making some amount of wash solution flow through thecolumn.
8. Centrifuge for 1 min. Transfer the Spin Column to a new, sterile
1.5 ml tube.
If the Spin Column has Buffer NW associated with it, centrifuge again for
additional 1 minute before transferring to the new 1.5 ml tube. Residualethanol from buffer NW may inhibit subsequent enzymatic reaction.

9. Apply 50µl of Buffer EB or dH2 O to the center of the membranein the Spin Column, let stand for 1 min, and centrifuge for 1 min.
Ensure that the Buffer EB or distilled water is dispensed directly onto thecenter of spin column membrane for optimal elution of DNA . To obtain moreconcentrated DNA solution, add 30 µl of Buffer EB or dH2 O. For long -term
storage, eluting in Buffer EB(10mM Tris-Cl, pH 8.5) or TE, pH 8.0 andstoring at -20℃ is recommended. When using water for elution, check thepH of water (>pH7.0) before elution.

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Troubleshooting guide
Low or no recovery
Buffer NW prepared incorrectly- Ensure that the appropriate volume of absolute ethanol printed on the bottle label
is added to Buffer NW.
Improper elution buffer- As user's requirement, elution buffer other than Buffer EB can be used. However,the conditions of optimal elution should be low salt concentration with alkaline pH(7<pH<9). When water or other buffer was used as eluent, ensure that conditions.
Elution buffer incorrectly dispensed- Ensure that elution buffer dispensed to the center of membrane. Incorrectly
dispensed elution buffer causes inappropriate contact with membrane, followed bypoor DNA recovery.
Gel slice incompletely solubilized- After addition of Buffer GB to the gel slice, mix by vortexing the tube every 2 –3
minutes during the 50℃ incubation. DNA will remain in any undissolved agarose.
Binding mixture turns orange or violet- The pH in the sample exceeds the buffer capacity of Buffer GB. Add 10 µl of 3 M
sodium acetate, pH 5.0, to the sample and mix. The color of the mixture will turnyellow indicating the correct pH for DNA binding. Even for binding mixtures with
only small color changes (slight orange color), add the 10µl sodium acetate.
Cloudy and gelatinous appearance of sample mixture after addition ofisopropanol-This may be due to salt precipitation, and will disappear upon mixing the sample.
Enzymatic reactions is not performed well with purified DNA
Salt concentration in eluate too high- Modify the wash step by incubating the spin column for 5 min at room
temperature after adding 750 µl of Buffer PE, then centrifuge

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DNA floats out of well while loading of agarose gel
Residual ethanol from Buffer NW remains in eluate- Discard flow-through from step 8, and then centrifuge for additional 2 min toremove ethanol completely before proceed to step 9.

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Contact us ;製造元

COSMO GENETECH CO.,Ltd.

Internet : www.cosmo4.com
E-mail : LaboPass@cosmo4.com
Tel : 82-2-922-7307
Fax : 82-2-922-7307

販売元
北海道システム・サイエンス株式会社

HokkaidoSystem Science.,Ltd.
北海道システム・サイエンス株式会社〒001-0932
札幌市北区新川西2条1丁目2-1
TEL : 011-768-5901
FAX : 011-768-5951
E-mail : bio@hssnet.co.jp
URL : http://www.hssnet.co.jp/

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