Simply Plant DNA Mini Kit
Explorer Ceramic & Steel
Manual
SG-P-028-1
Contents
Contents
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General Information
Introduction . . . . . . . .
Intended use . . . . . . .
Product specifications . . .
List of Components . . . .
Storage conditions . . . .
Safety information . . . . .
Hazard Identification
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Additional Requirements
Required laboratory equipment and additives . . . . . . . . . . .
Required components not included . . . . . . . . . . . . . . . . .
Additional Information . . . . . . . . . . . . . . . . . . . . . . . .
Preselection of the most suitable homogenization procedure
Preselection of the most suitable lysis solution . . . . . . . .
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Protocol
Recommended steps before using this kit . . . . . . . . .
Homogenization Procedure . . . . . . . . . . . . . . . . .
Disruption of plant material by bead mill homogenizer
Disruption of plant material by liquid nitrogen . . . .
Purification Procedure . . . . . . . . . . . . . . . . . . .
DNA Isolation from plant using Lysis Solution B . . .
DNA Isolation from plant using Lysis Solution C . . .
DNA Isolation from plant using Lysis Solution D . . .
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Troubleshooting
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Warranties and Quality Control
15
6
Technical support
Legal Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 1
1
General Information
Introduction
The Simply Plant DNA Mini Kit Explorer Ceramic & Steel provides a toolkit to explore
a fast and simple method for the preparation of genomic DNA from different kinds of plant
materials, according to your requirements.
The Simply Plant DNA Mini Kit Explorer Ceramic & Steel ensures optimal processing,
purification performance, high yield and excellent DNA quality for the most common plant
species. You can choose between different homogenization-methods (steel- or ceramicbeads or liquid nitrogen) and three different lysis solutions. This kit enables you to find an
optimal preparation method adapted to your plant material. The extraction procedure is
based on binding nucleic acids to a surface of a Spin Filter membrane and consists of four
steps. The first step is the homogenization of the starting material. Afterwards the DNA
binds to a Spin Filter membrane, washed and, finally, eluted.
Intended use
The Simply Plant DNA Mini Kit Explorer Ceramic & Steel is designed for the preparation of genomic DNA from different kinds of plant materials (leafs, fruits, woods, needles
and seeds). It is able to process up to 100 mg (dry weight) or 180 mg (wet weight) of
starting material, regardless of whether it is fresh or frozen. The extracted DNA is suitable
for a wide range of different molecular biology downstream applications, including PCR,
restriction digestion, cloning and sequencing.
B
Page 2
For Research Use Only — Not for use in diagnostic procedures.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Product specifications
Starting Material
Fresh, frozen (up to 180 mg wet weight) or dried plant material (up to
100 mg dry weight)
Time for preparation
30 - 40 minutes
Binding capacity
approximately 50 µg DNA
Expected Yield
3 - 25 µg DNA, depending on the kind and quantity of plant material
Order number
SG-P-028-1 (50 reactions)
Manual version
SG-P-028-1-1
List of Components
Component
Quantity
Storage
Cat No
2 ml Tube (50 Tubes)
1 pcs
15 - 25°C
SG-C-065-1
Binding Solution A
15 ml
15 - 25°C
SG-C-061-1
Ceramic Beads (25 Tubes)
1 pcs
15 - 25°C
SG-C-084-2
250 pcs
15 - 25°C
SG-C-009-1
Elution Buffer A
15 ml
15 - 25°C
SG-C-063-3
Elution Tube A
50 pcs
15 - 25°C
SG-C-038-1
Lysis Solution B
25 ml
15 - 25°C
SG-C-053-1
Lysis Solution C
25 ml
15 - 25°C
SG-C-054-1
Lysis Solution D
25 ml
15 - 25°C
SG-C-055-1
Precipitation Buffer A
4x 2 ml
15 - 25°C
SG-C-033-1
Prefilter A
50 pcs
15 - 25°C
SG-C-010-1
Proteinase K II
1 pcs
4°C (if dissolved: -20°C)
SG-C-022-1
Spin Filter Green A
50 pcs
15 - 25°C
SG-C-012-1
Steel Beads (25 Tubes)
1 pcs
15 - 25°C
SG-C-085-2
Washing Solution D
15 ml
15 - 25°C
SG-C-059-1
Collection Tube B
Storage conditions
All components of the Simply Plant DNA Mini Kit Explorer Ceramic & Steel except for
the Proteinase K II should be stored dry at room temperature (15 to 25°C).
Lyophilized Proteinase K should be stored at 4°C. Dissolved Proteinase K II should be
stored at -20°C. Repeated freezing and thawing should be avoided. Allocation of the solution into aliquots is recommended.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 3
If there are any precipitates within the provided solutions solve these precipitates by careful
warming. Do not use the kit after the expiration date.
Safety information
All reagents in this kit have to be used for the intended use described above only. The kit
should be used by authorized staff only. It is strongly recommended to follow the instruction for use. Inappropriate handling or use of the kit or non-observance of the instructions
for use can increase the danger for your health and reduce the reliability of results. The
reagents should be stored inside the original vessels at the given storage temperatures as
indicated on the Fact Sheet. Single components of different lots and consumables should
not be exchanged. The expiry dates have to be considered. Please pay attention to the
regulations for the prevention of accidents in laboratories. In particular, the following safety
precautions have to be considered:
B
Don’t eat, drink or smoke! Always wear protective clothing, safety glasses and
gloves! Do not add bleach or acidic components to the waste after sample
preparation! All liquid waste must be considered as potentially infectious and
must be handled and discarded according to local safety regulation!
For more information, please read the Fact Sheet and ask for the material safety data
sheets (MSDS’s). Emergency medical information in English and German can be obtained 24 hours a day from the Poison Information Center in Freiburg (Germany), Phone:
+49 761 19240.
Hazard Identification
Please read carefully the following hazard identification information that classifies the substances or mixtures contained in this product.
Proteinase K II
Signal word
DANGER
Pictograms
Hazard statement(s)
H315
Causes skin irritation.
H319
Causes serious eye irritation.
H334
May cause allergy or asthma symptoms or breathing difficulties if inhaled.
Continued on next page
Page 4
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
continued from previous page
H335
May cause respiratory irritation.
Precautionary statement(s)
P261
Avoid breathing dust/fume/gas/mist/vapours/spray.
P305 + P351 + P338
IF IN EYES: Rinse cautiously with water for several minutes. Remove
contact lenses, if present and easy to do. Continue rinsing.
P342 + P311
If experiencing respiratory symptoms: Call a POISON CENTER / doctor / ...
R-phrase(s)
R32
Contact with acids liberates very toxic gas.
R36/37/38
Irritating to eyes, respiratory system and skin.
S-phrase(s)
S22
Do not breathe dust.
S24
Avoid contact with the skin.
S26
In case of contact with eyes, rinse immediately with plenty of water
and seek medical advice.
S36/37
Wear suitable protective clothing and gloves.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 5
2
Additional Requirements
Required laboratory equipment and additives
2 Bead mill homogenizer
2 Microcentrifuge
2 Thermomixer
2 Vortexer
2 Variable pipettes for 10 µl – 1 000 µl
2 Sterile pipette tips with protection against contamination
Required components not included
2 optionally RNase A (100 mg/ml)
2 ddH2 O
2 96 - 99.8 % ethanol
Page 6
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Additional Information
Preselection of the most suitable homogenization procedure
Complete and quick disruption of starting material is essential to ensure high DNA yields
and to avoid DNA degradation. The lysis procedure is most effective with well-homogenized,
powdered samples. The following homogenization procedures are recommended for different kinds of plant material:
Disruption of plant material by bead mill homogenizer
A suitable method is the homogenization with any type of commercial homogenizers or
bead mills using ceramic or steel beads. Choose the optimal kind of beads (included in
this kit) depending on your starting material:
• Ceramic beads (2.4 – 2.6 mm): For soft plant material and leaves.
• Steel beads (4.7 mm): For seeds, rice and needles.
Disruption of plant material by liquid nitrogen
The homogenization of any kind of plant material with a mortar and pestle in the presence of liquid nitrogen is a classical and well established method. It is compatible with all
following protocols for DNA preparation.
Preselection of the most suitable lysis solution
Plant parts consist of very different materials and contain various amounts of polyphenols,
acidic components, or polysaccharides that can lead to a decrease of the extracted DNA
yield or to trouble in downstream applications. This kit contains three different lysis solutions to enable you to find the optimal solution and adjust the DNA preparation to your
specific conditions in the laboratory. You will get high yields and an excellent quality of
DNA for most plant species. Below the lysis solutions are listed:
• Lysis Solution B: This solution has been optimized for genomic DNA isolation from
seeds, but can also be used for other plant materials. It requires subsequent precipitation with Precipitation Buffer A to remove all contaminations.
• Lysis Solution C: This solution contains SDS (sodium dodecyl sulfate) and requires
subsequent precipitation with Precipitation Buffer A to remove all contaminations.
• Lysis Solution D: This solution contains the detergent CTAB (cetyltrimethylammonium bromide) and can be used for a standard protocol.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 7
In order to identify the optimal conditions for the lysis of different plant materials, we propose to use one batch of the same starting material and compare all three lysis buffer
simultaneously. The decision for the most effective lysis buffer should be made regarding
yield and quality of the extracted genomic DNA. For some plant species Lysis solution C
and B might be used with similar results. In these cases please make a choice for the
easiest protocol.
B
Page 8
For high yield and excellent DNA quality combine the selected
homogenization procedure with the most suitable lysis solution!
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
3
Protocol
This protocol is valid for the preparation of genomic DNA from different kinds of plant material (leafs, fruits, woods, needles as well as seeds). It is able to process up to 100 mg (dry
weight) or 180 mg (wet weight) of starting fresh or frozen material. Choose the protocol of
your previously selected homogenization procedure and lysis solution.
Recommended steps before using this kit
Before first use:
2 Add 56 ml of ethanol (96 – 99.8 %)
2 Add 1.5 ml of ddH2 O, mix thoroughly and store in aliquots at -20°C
Before use:
• Homogenization: Choose the optimal homogenization procedure for your plant material (see Additional Information). For homogenization using liquid nitrogen, precooling of mortar, pestle and tubes for sample storage is recommended.
• Lysis Solution: Choose the optimal lysis solution for your plant material (see Additional Information).
• Thermomixer: Preheat to 65°C
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 9
Homogenization Procedure
Disruption of plant material by bead mill homogenizer
1. Choose the optimal kind of beads depending on your starting material. For soft
plant material and leaves use Ceramic Beads (2.4 – 2.6 mm) and for seeds, rice and
needles use Steel Beads (4.7 mm).
2. Place 50 – 100 mg (120 - 180 mg wet weight) of starting material into a bead
tube (ceramic or steel, depending on starting material) and add 50 µl of ddH2 O.
3. Vortex for 30 seconds using the bead mill homogenizer to homogenize the plant
material. Repeat the procedure until the entire plant material is milled to a fine
solution.
4. Transfer the homogenized starting material into a 2 ml Tube.
5. Proceed immediately to Purification according to your previously selected lysis solution.
Disruption of plant material by liquid nitrogen
1. Freeze 50 – 100 mg (120 - 180 mg wet weight) of starting material in liquid nitrogen and avoid thawing the sample at any time to homogenize the plant material.
2. Pulverize frozen plant material to a fine powder.
3. Refill the mortar with liquid nitrogen to keep the material frozen.
4. Transfer the finely crushed plant material into a precooled 2 ml Tube. Make sure all
liquid nitrogen has evaporated before closing the tube.
5. Proceed immediately to Purification according to your previously selected lysis solution.
Page 10
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Purification Procedure
DNA Isolation from plant using Lysis Solution B
• Add 400 µl of Lysis Solution B and 20 µl Proteinase K II to the sample. Mix by
pulsed vortexing for 5 seconds.
• Incubate at 65°C for approximately 30 - 60 minutes at 550 rpm.
• Add 100 µl of Precipitation Buffer A and vortex shortly for 5 seconds.
• Incubate at room temperature for 5 minutes and centrifuge at maximum speed
(above 10 000 x g) for 5 minutes.
• Place the Prefilter A into a Collection Tube B.
• Apply the sample to the Prefilter A. Centrifuge at 10 000 x g for 1 minute.
• Discard the Prefilter A and keep working with the filtrate in the Collection Tube. Don’t
discard the Collection Tube B with the filtrate!
Note: Add 4 µl of RNase A stock solution (100 mg/ml) to remove RNA from
sample, vortex shortly and incubate for 5 minutes at RT.
• Add 200 µl of Binding Solution A to the filtrate. Mix carefully by pipetting up and
down several times to get a homogenous solution. Don’t vortex!
• Place the Spin Filter Green A into a Collection Tube B.
• Apply the sample to the Spin Filter Green A and close the cap. Centrifuge at
10 000 x g for 2 minutes.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Centrifuge at maximum speed (above 10 000 x g) for 2 minutes to avoid a carry-over
of ethanol residuals.
• Place the Spin Filter Green A into the 1.5 ml Elution Tube A.
• Add 100 – 200 µl of Elution Buffer A and incubate at room temperature for 1 minute.
Centrifuge at 6 000 x g for 1 minute.
Note: To increase the concentration of the extracted DNA, elute in two steps with
Elution Buffer A (e.g. 100 µl + 100 µl) or elute with volumes smaller than 200 µl.
• Store the extracted DNA at 4°C. Long-term storage of DNA should happen at -20°C.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 11
DNA Isolation from plant using Lysis Solution C
• Add 400 µl of Lysis Solution C and 20 µl Proteinase K II to the sample. Mix by
pulsed vortexing for 5 seconds.
• Incubate at 65°C for approximately 30 - 60 minutes at 550 rpm.
• Add 100 µl of Precipitation Buffer A and vortex shortly for 5 seconds.
• Incubate at room temperature for 5 minutes and centrifuge at maximum speed
(above 10 000 x g) for 5 minutes.
• Place the Prefilter A into a Collection Tube B.
• Apply the sample to the Prefilter A. Centrifuge at 10 000 x g for 1 minute.
• Discard the Prefilter A and keep working with the filtrate in the Collection Tube B.
Don’t discard the Collection Tube B with the filtrate!
Note: Add 4 µl of RNase A stock solution (100 mg/ml) to remove RNA from
sample, vortex shortly and incubate for 5 minutes at RT.
• Add 200 µl of Binding Solution A to the filtrate. Mix carefully by pipetting up and
down several times to get a homogenous solution. Don’t vortex!
• Place the Spin Filter Green A into a Collection Tube B.
• Apply the sample to the Spin Filter Green A and close the cap. Centrifuge at
10 000 x g for 2 minutes.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Centrifuge at maximum speed (above 10 000 x g) for 2 minutes to avoid a carry-over
of ethanol residuals.
• Place the Spin Filter Green A into the 1.5 ml Elution Tube A.
• Add 100 – 200 µl of Elution Buffer A and incubate at room temperature for 1 minute.
Centrifuge at 6 000 x g for 1 minute.
Note: To increase the concentration of the extracted DNA, elute in two steps with
Elution Buffer A (e.g. 100 µl + 100 µl) or elute with volumes smaller than 200 µl.
• Store the extracted DNA at 4°C. Long-term storage of DNA should happen at -20°C.
Page 12
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
DNA Isolation from plant using Lysis Solution D
• Add 400 µl of Lysis Solution D and 20 µl Proteinase K II to the sample. Mix by
pulsed vortexing for 5 seconds.
• Incubate at 65°C for approximately 30 - 60 minutes at 550 rpm.
• Place the Prefilter A into a Collection Tube B.
• Apply the sample to the Prefilter A and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Discard the Prefilter A and keep working with the filtrate in the Collection Tube B.
Don’t discard the Collection Tube B with the filtrate!
Note: Add 4 µl of RNase A stock solution (100 mg/ml) to remove RNA from
sample, vortex shortly and incubate for 5 minutes at RT.
• Add 200 µl of Binding Solution A to the filtrate. Mix carefully by pipetting up and
down several times to get a homogenous solution. Don’t vortex!
• Place the Spin Filter Green A into a Collection Tube B.
• Apply the sample to the Spin Filter Green A and close the cap. Centrifuge at
10 000 x g for 2 minutes.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Add 650 µl of Washing Solution D and close the cap. Centrifuge at 10 000 x g for
1 minute.
• Place the Spin Filter Green A into a new Collection Tube B and discard the used
Collection Tube B.
• Centrifuge at maximum speed (above 10 000 x g) for 2 minutes to avoid a carry-over
of ethanol residuals.
• Place the Spin Filter Green A into the Elution Tube A.
• Add 100 – 200 µl of Elution Buffer A and incubate at room temperature for 1 minute.
Centrifuge at 6 000 x g for 1 minute.
Note: To increase the concentration of the extracted DNA, elute in two steps with
Elution Buffer A (e.g. 100 µl + 100 µl) or elute with volumes smaller than 200 µl.
• Store the extracted DNA at 4°C. Long-term storage of DNA should happen at -20°C.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 13
4
Troubleshooting
Problem
Probable Cause
Remedy
Increase lysis time
Slow flow speed through
the Spin Filter Green A
Insufficient lysis
Increase centrifugation speed / time
Overloading of Spin Filter Green A
reduces yield
Too much starting material
Reduce amount of starting material
Insufficient lysis
See above
Insufficient mixing with Binding
Solution A
Mix carefully by pipetting up and down
3 - 4 times. Don’t vortex!
Poor DNA yield
Incomplete elution
Increase the incubation time with Elution
Buffer A to 5 minutes
Repeat elution step once again
Increase the Elution Buffer A volume
Degraded or sheared
DNA
Too much Elution Buffer A
Elute the DNA with lower Elution Buffer A
volume
Incorrect storage of starting
material
Ensure that starting material is frozen
immediately in liquid nitrogen or minimum
at -20°C and stored continuously at
-80°C.
Avoid thawing and freezing
Low performance of
downstream applications
RNA contamination
Page 14
Old material
Old material often contains degraded DNA
Incorrect storage of the DNA
Avoid thawing and freezing
Salt carry-over
Ensure proper washing
Check Washing Solution D for salt
precipitates
Ethanol carry-over
Increase centrifugation time for ethanol
removal
RNA
Digest RNA with RNase
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
5
Warranties and Quality Control
Simply Genomics guarantees the correct performance of each kit for applications as described in the instruction for use.
The purchaser must determine the suitability of the product for its particular use.
We reserve the right to change, alter or modify any product to enhance its performance
and design.
If a Simply Genomics product does not meet your expectations or if you are unsatisfied
for any reason with this product, please contact our Technical Assistance.
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Page 15
6
Technical support
If you have any questions regarding any aspects of this product, please do not hesitate to
contact us via
[email protected]
or call us at +49 (0) 3641 / 777 888.
Legal Information
This product has been manufactured, distributed and published by:
Moldiax GmbH
Konrad-Zuse-Str. 1
07745 Jena
Germany
Phone: +49 (0) 3641 / 777 333
E-Mail: [email protected]
Internet: www.moldiax.de
© Copyright 2014, Moldiax GmbH
Page 16
Simply Plant DNA Mini Kit Explorer Ceramic & Steel
Moldiax GmbH
Konrad-Zuse-Straße 1
07745 Jena / Germany
Telefon +49 (0) 36 41 / 777 333
Telefax +49 (0) 36 41 / 777 67 333
[email protected]
www.simplygenomics.com