PRODUCT INFORMATION

Thermo Scientific
GeneJET Gel Extraction Kit
#K0691, #K0692

www.thermoscientific.com/onebio

#__

Lot __
CERTIFICATE OF ANALYSIS

The kit was tested in the extraction of 100 bp DNA fragment from a 2% agarose gel and a 5.5 kb
DNA fragments from 1% agarose gel according to the protocol described in the manual. The
quality of the extracted DNA was evaluated spectrophotometrically, by agarose gel
electrophoresis, digestion with Thermo Scientific FastDigest restriction enzymes and automated
fluorescent sequencing.
Quality authorized by:

Rev.6.

hhhh

Jurgita Zilinskiene

CONTENTS

page

COMPONENTS OF THE KIT ................................................................. 2

STORAGE AND STABILITY .................................................................. 2
DESCRIPTION....................................................................................... 2
PRINCIPLE ............................................................................................ 3
IMPORTANT NOTES ............................................................................. 3
ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED .................. 3
PURIFICATION PROTOCOL ................................................................. 4
TROUBLESHOOTING ........................................................................... 6
SAFETY INFORMATION ....................................................................... 8

COMPONENTS OF THE KIT

GeneJET Gel Extraction Kit
Binding Buffer
Wash Buffer (concentrated)
Elution Buffer (10 mM Tris-HCl, pH 8.5)
GeneJET Purification Columns (preassembled
with collection tubes)

50 preps
#K0691

250 preps
#K0692

30 ml
9 ml
15 ml

150 ml
45 ml
30 ml

50

250

STORAGE AND STABILITY

The Thermo Scientific GeneJET Gel Extraction Kit can be stored for up to 12 months at room
temperature (15-25C) or at 4C for storage periods longer than 12 months. Any precipitate in
the buffers can be re-dissolved by incubating at 37C before use.
DESCRIPTION

The GeneJET Gel Extraction Kit is designed for rapid and efficient purification of DNA fragments

from standard or low-melting point agarose gels run in either TAE or TBE buffer.
The kit utilizes a proprietary silica-based membrane technology in the form of a convenient
spin column. The kit can be used to purify DNA fragments from 25 bp to 20 kb in size. The
recovery rates are up to 95% in a 100 bp 10 kb DNA fragment size range (see Fig. 1). Each
GeneJET purification column has a binding capacity of up to 25 g of DNA and can process
up to 1 g of agarose gel.
The entire procedure takes just 15 min and the isolated DNA is ready to use in all common
downstream applications including ligation, restriction digestion, PCR, sequencing and
labeling.

Fig. 1. Recovery dependence on DNA fragment size

PRINCIPLE
The DNA fragment of interest is excised from an agarose gel, placed in a microcentrifuge tube,
solubilized in binding buffer and applied to the column. The chaotropic agent in the binding
buffer dissolves agarose, denatures proteins and promotes DNA binding to the silica
membrane in the column. As an added convenience, the binding buffer contains a color
indicator that allows for easy monitoring of the solution pH for optimal DNA binding. Impurities
are removed with a simple wash step. Purified DNA is then eluted from the column with the
elution buffer. The recovered DNA is ready for use in downstream applications.
IMPORTANT NOTES
Prior to the initial use of the kit, dilute the Wash Buffer (concentrated) with ethanol
(96-100%):
50 preps
250 preps
#K0691
#K0692
Wash Buffer (concentrated)
9 ml
45 ml
Ethanol
45 ml
225 ml
Total Volume
54 ml
270 ml
After the ethanol has been added, mark the check box on the bottle to indicate the
completed step.
Examine the Binding Buffer for precipitates before each use. Re-dissolve any precipitate
by warming the solution to 37C and cooling to 25C.
Wear gloves when handling the Binding Buffer as this solution contains irritants (see p.7
for SAFETY INFORMATION).
Do not reuse electrophoresis buffer when extracted DNA fragment will be used directly for
sequencing.

ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED

Ethanol 96-100%.
Isopropanol.
3 M sodium acetate, pH 5.2 (may be necessary).
Microcentrifuge.
1.5 or 2 ml microcentrifuge tubes.
Heating block or water bath.

PURIFICATION PROTOCOL
Note
Read IMPORTANT NOTES on p. 3 before starting.
All purification steps should be carried out at room temperature.
All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g
(10 000-14 000 rpm, depending on the rotor type).
Step

Procedure
Excise gel slice containing the DNA fragment using a clean scalpel or razor
blade. Cut as close to the DNA as possible to minimize the gel volume. Place
the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the
gel slice.

Note. If the purified fragment will be used for cloning reactions, avoid damaging the DNA
through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a
glass or plastic plate during UV illumination.

Add 1:1 volume of Binding Buffer to the gel slice (volume: weight)
(e.g., add 100 l of Binding Buffer for every 100 mg of agarose gel).

Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer
to the gel slice.

Incubate the gel mixture at 50-60C for 10 min or until the gel slice is
completely dissolved. Mix the tube by inversion every few minutes to facilitate
the melting process. Ensure that the gel is completely dissolved. Vortex the gel
mixture briefly before loading on the column.
Check the color of the solution. A yellow color indicates an optimal pH for DNA
binding. If the color of the solution is orange or violet, add 10 l of 3 M sodium
acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

4
for 500
bp and
>10 kb
DNA
fragments

Optional: use this step only when DNA fragment is <500 bp or >10 kb long.
If the DNA fragment is <500 bp, add a 1:2 volume of 100% isopropanol to
the solubilized gel solution (e.g. 100 l of isopropanol should be added to
100 mg gel slice solubilized in 100 l of Binding Buffer). Mix thoroughly.
If the DNA fragment is >10 kb, add a 1:2 volume of water to the solubilized
gel solution (e.g. 100 l of water should be added to 100 mg gel slice
solubilized in 100 l of Binding Buffer). Mix thoroughly.

Transfer up to 800 l of the solubilized gel solution (from step 3 or 4) to the

GeneJET purification column. Centrifuge for 1 min. Discard the flow-through
and place the column back into the same collection tube.

Note. If the total volume exceeds 800 l, the solution can be added to the column in stages.
After each application, centrifuge the column for 30-60 s and discard the flow-through after
each spin. Repeat until the entire volume has been applied to the column membrane. Do not
exceed 1 g of total agarose gel per column.

Step

Procedure

Optional: use this additional binding step only if the purified DNA will be used for
sequencing.
Add 100 l of Binding Buffer to the GeneJET purification column. Centrifuge
for 1 min. Discard the flow-through and place the column back into the same
collection tube.

Add 700 l of Wash Buffer (diluted with ethanol as described on p. 3) to the

GeneJET purification column. Centrifuge for 1 min. Discard the flow-through
and place the column back into the same collection tube.

Centrifuge the empty GeneJET purification column for an additional 1 min to

completely remove residual wash buffer.
Note. This step is essential to avoid residual ethanol in the purified DNA solution. The
presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

Transfer the GeneJET purification column into a clean 1.5 ml microcentrifuge

tube (not included). Add 50 l of Elution Buffer to the center of the purification
column membrane. Centrifuge for 1 min.
9

10

Note
For low DNA amounts the elution volumes can be reduced to increase DNA
concentration. An elution volume between 20-50 l does not significantly reduce the
DNA yield. However, elution volumes less than 10 l are not recommended.
If DNA fragment is >10 kb, prewarm Elution Buffer to 65C before applying to column.
If the elution volume is 10 l and DNA amount is 5 g, incubate column for 1 min at
room temperature before centrifugation.

Discard the GeneJET purification column and store the purified DNA at -20C.

TROUBLESHOOTING
Problem
Low DNA yield

Possible Cause and Solution

Incomplete solubilization of the gel slice
Verify that a 1:1 volume of Binding Buffer is added to a precisely
weighted gel slice (e.g. for every 100 mg of agarose gel, add 100 l
of Binding Buffer).
Ensure that the gel slice is completely dissolved before applying to
the GeneJET purification column. A large amount of agarose or a gel
slice with an agarose percentage greater than 2% may require extra
time to dissolve. In some cases larger volumes of Binding Buffer and
additional vortexing of the gel solution facilitate solubilization.
Inefficient DNA binding
Check the color of the solution after the gel slice is completely
dissolved. A yellow color indicates an optimal pH for DNA binding. If
the solution color is orange or violet, add 10 l of 3 M sodium
acetate, pH 5.2 solution and mix. The color of the mix will become
yellow.
Inefficient membrane wash
Ensure that recommended volume of ethanol was added to the Wash
Buffer (concentrated) prior first use (see p. 3).
Inefficient DNA elution
Add the Elution Buffer directly to the center of the membrane and not
to the side of the GeneJET purification column.
Use 20-50 l of Elution Buffer and ensure that the volume completely
covers the surface of the membrane.
Increase the Elution Buffer volume twice or perform two elution cycles
when purifying larger amounts of DNA (e.g. >15 g).
In step 8, ensure all residual wash buffer is removed from the
membrane. Longer centrifugation time (extra minute) can aid in
removal of wash buffer.

DNA does not

remain in an
agarose gel well

Presence of residual ethanol

In step 8, ensure all residual wash buffer is removed from the
membrane. Longer centrifugation time can aid in removal of wash
buffer.

Low quality
sequencing
results

Contamination from reused electrophoresis buffer

If extracted DNA will be used directly for sequencing, freshly prepared
electrophoresis buffers should be used both for gel preparation and
for gel running.

Problem

Possible Cause and Solution

Downstream
applications are
unsuccessful

Presence of residual ethanol

In step 8, ensure all residual wash buffer is removed from the
membrane. A prolonged centrifugation time can aid in removal of
wash buffer.
Inefficient membrane wash
If the collection tube is overfilled during the wash step, some of the
wash buffer may remain in the bottom of the GeneJET purification
column. To avoid this, always discard the flow-through after
centrifugation.
Eluate contaminated with agarose
Ensure the gel slice is properly solubilized during steps 1-4. Verify
that a 1:1 volume of Binding Buffer was added to a precisely
weighted gel slice. Large amounts of agarose or agarose gel
percentages greater than 2% may take more time to dissolve. In
some cases adding a larger volume of Binding Buffer and vortexing
the gel solution more frequently can facilitate solubilization.
Eluate contaminated with excess salt
Ensure that the wash in step 7 is effecitve. Incubate the GeneJET
purification column with the Wash Buffer for several minutes before
proceeding to centrifugation.

References
1. Vogelstein, B. and Gillespie, D., Preparative and analytical purification of DNA from agarose,
Proc. Natl. Acad. Sci. USA, 76, 615-619, 1979.
2. Marko, M.A., Chipperfield, R. and Birnboim, H.C., A procedure for the large-scale isolation of
highly purified plasmid DNA using alkaline extraction and binding to glass powder, Anal.
Biochem., 121, 382-387, 1982.
3. Boom, R., Sol, C.J.A., et al., Rapid and simple method for purification of nucleic acids, J.
Clin. Microbiol., Mar, 495-503, 1990.

SAFETY INFORMATION

Xn Harmful

Binding Buffer
Hazard-determining component of labeling: guanidinium thiocyanate

Risk phrases
R20/21/22 Harmful by inhalation, in contact with skin and if swallowed.
R32
Contact with acids liberates very toxic gas.
R52/53
Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic
environment.
Safety phrases
S9
Keep container in a well-ventilated place.
S23
Do not breathe gas/fumes/vapour/spray.
S36/37
Wear suitable protective clothing and gloves.
S60
This material and its container must be disposed of as hazardous waste.
S61
Avoid release to the environment. Refer to special instructions/safety data sheets.

PRODUCT USE LIMITATION

This product is developed, designed and sold exclusively for research purposes and in vitro use only. The
product was not tested for use in diagnostics or for drug development, nor is it suitable for administration to
humans or animals.
Please refer to www.thermoscientific.com/onebio for Material Safety Data Sheet of the product.
2012 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher
Scientific Inc. and its subsidiaries.
11