Omni 26-022 User manual

PLANT DNA PURIFICATION KIT
User Manual
26-022............5 preps
26-023............50 preps
26-023B.........50 preps
P/N 0329310 REV. B

Plant DNA Purification Kit
1. Introduction.....................................................................Pg. 1
2. Overview............................................................................Pg. 1
3. Kit Contents ...................................................................Pg. 2
4. Storage and Stability ..................................................Pg. 3
5. Illustrated Protocols ....................................................Pg. 3
6. Before Starting................................................................Pg. 4
7. Preparing Reagents.....................................................Pg. 4
8. Plant DNA Kit Directions ..........................................Pg. 5
10. Trouble Shooting Guide........................................Pg. 7

Plant DNA Purification Kit
INTRODUCTION
The Omni Plant DNA Purification Kit is designed for efficient recovery of
genomic DNA up to 40 kb in size from fresh, frozen, or dried plant tissue
samples rich in polysaccharides or having a lower DNA content. Up to 100
mg wet tissue (or 30 mg dry tissue) can be processed in less than 1 hour.
The system combines the reversible nucleic acid-binding properties of the
Omni Mini DNA Column matrix with the speed and versatility of spin column
technology to eliminate polysaccharides, phenolic compounds, and enzyme
inhibitors from plant tissue lysates. Purified DNA is suitable for PCR, restriction
digestion, and hybridization applications.
OVERVIEW
If using the Omni Plant DNA Purification Kit for the first time, please read this
booklet to become familiar with the procedures. This procedure relies on the
well-established properties of the cationic detergent, cetyltrimethyl ammonium
bromide (CTAB), in conjunction with the selective DNA binding of the Omni Mini
DNA Column matrix, to isolate high-quality DNA. Samples are homogenized
and lysed in a high salt buffer containing CTAB and extracted with chloroform
to remove polysaccharides and other components that interfere with many
routine DNA isolations and downstream applications. Binding conditions are
adjusted, and DNA is purified using an Omni DNA Mini Column. Salts, proteins,
and other contaminants are removed to yield high-quality genomic DNA
suitable for downstream applications such as endonuclease digestion, thermal
cycle amplification, and hybridization applications.
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Plant DNA Purification Kit
2
KIT CONTENTS
Product Number 26-022 26-023 26-023B
Purications 5 50 50
Omni DNA Mini Columns 5 50 50
2 mL Collection Tubes 10 100 100
BB Buer 5 mL 20 mL 20 mL
DW Buer 1.5 mL 20 mL 20 mL
EB Buer 2 mL 30 mL 30 mL
CTB Buer 5 mL 40 mL 40 mL
Antifoam 1 mL 1 mL
2 mL Bead Kit 2.8 mm ceramic 5 50
User Manual
STORAGE AND STABILITY
All of the Omni Plant DNA Purification Kit components are guaranteed
for at least 12 months from the date of purchase when stored at room
temperature. During shipment or storage in cool ambient conditions,
precipitates may form in CTB Buffer and BB Buffer. Dissolve such deposits
by warming the solution at 37°C and gently shaking.

Plant DNA Purification Kit
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ILLUSTRATED PROTOCOLS
Lyse and homogenize
sample
Remove polysaccherides
and polyphenols
Adjust the
binding conditions
Transfer lysate into a
DNA mini column
to bind DNA
Wash column x 2
Dry the column
Elute DNA

Plant DNA Purification Kit
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BEFORE STARTING
This method is suitable for the isolation of DNA from up to 100 mg of fresh/
frozen plant tissue and up to 30 mg of dried plant tissue.
Yields vary depending on source.
Materials and equipment to be supplied by user:
• Tabletop microcentrifuge capable of 10,000 x g
• Nuclease-free 1.5 mL microcentrifuge tubes
• Waterbath or incubator capable of 65ºC
• Bead Mill Homogenizer or vortexer
• β-mercaptoethanol (βME)
• Chloroform and isoamyl alcohol (24:1)
• Optional: RNase stock solution (20 mg/mL)
• 100% Ethanol
PREPARING REAGENTS
1. Dilute DW Buffer with 100% ethanol as follows and store at room temperature.
Kit 100% Ethanol to be added
26-022 6 mL
26-023 and 26-023B 80 mL
2. Prepare or obtain a mixture of chloroform: isoamyl alcohol (24:1)
3. β-mercaptoethanol (βME) must be added to CTB Buffer before use
(20 µL βME/1 mL CTB).

Plant DNA Purification Kit
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5. Add 700 μL chloroform/isoamyl alcohol (24:1). Vortex vigorously to mix.
6. Centrifuge at ≥10,000 x g for 5 minutes,
7. Transfer 300 μL aqueous phase (top) to a new microcentrifuge tube,
making sure not to disturb the pellet or transfer any debris.
8. Add 150 μL BB Buffer and 300 μL 100% ethanol. Vortex to obtain a
homogeneous mixture.
NOTE: A precipitate may form upon addition of ethanol; it will not interfere
with DNA isolation.
PLANT DNA PURIFICATION KIT DIRECTIONS
26-023 - Tissue Digestion
1. Mince up to 100 mg fresh/frozen
plant tissue
(up to 30 mg dried tissue)
2. Transfer to a clean 1.5 mL
microcentrifuge tube
(not provided)
3. Add 500 µL CTB Buffer. Vortex
vigorously to mix. Make sure to
disperse all clumps.
NOTE: Ensure βME is added to CTB
Buffer (20 µL βME/1 mL CTB).
Optional: Add 5 µL RNase. Let sit
at room temperature for 5 minutes.
Proceed to step 4.
4. Incubate at 65°C for 30 minutes.
Invert the samples twice
during incubation.
26-023B - Tissue Homogenization
1. Weigh up to 100 mg of fresh/
frozen plant tissue (up to 30 mg
dried tissue) and transfer to the
2 mL tube containing 2.8mm
ceramic beads.
2. Add 500 µL of CTB buffer and
10 µL of Antifoam.
NOTE: Ensure βME is added to CTB
Buffer (20 µL βME/1 mL CTB).
3. Dissociate sample on a bead mill
at 3.5 – 4 m/s for 20 seconds or
with continuous vortexing for
10 minutes.
Optional: After homogenization,
add 5 µL RNase A. Let sit at room
temperature for 5 minutes.
Proceed to step 4.
4. Incubate at 65°C for 15 minutes.
Invert the samples twice
during incubation.

Plant DNA Purification Kit
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9. Insert an Omni DNA Mini Column into a 2 mL Collection Tube.
10.Transfer the entire sample (including any precipitate that may have
formed) to the Omni DNA Mini Column.
11.Centrifuge at 10,000 x g for 1 minute.
12.Discard the filtrate and the Collection Tube.
13.Transfer the Omni DNA Mini Column to a new 2 mL Collection Tube.
14.Add 700 μL DW Buffer.
NOTE: DW Buffer must be diluted with 100% ethanol prior to use.
15.Centrifuge at 10,000 x g for 1 minute.
16.Discard the filtrate and reuse the Collection Tube.
17.Repeat Steps 14-16 for a second DW Buffer wash step.
18.Centrifuge the empty Omni DNA Mini Column at maximum speed
for 2 minutes to dry the membrane.
NOTE: It is critical to remove any trace of ethanol that may otherwise
interfere with downstream applications.
19.Transfer the Omni DNA Mini Column to a nuclease-free 1.5 or 2 mL
microcentrifuge tube (not provided).
20.Add 50 μL EB Buffer (or sterile deionized water) heated to 65°C.
21.Centrifuge at 10,000 x g for 1 minute.
22.Repeat Steps 20-21 for a second elution step.
23.Store DNA at -20°C.

Plant DNA Purification Kit
Problem Cause Suggestion
Clogged Column
Carry-over of debris.
Following extraction with
chloro:isoamyl alcohol, make
sure no particulate material is
transferred.
Sample too viscous.
Do not exceed suggested
amount of starting material.
Alternatively, increase
amounts of Buers CTB and
BB and use two or more
columns per sample.
Low DNA yield
Incomplete disruption of
starting material.
For both dry and fresh
samples, obtain a ne
homogeneous powder before
adding CTB Buer or use a
Bead Ruptor if possible.
Poor lysis of sample
Decrease amount of starting
material or increase amount
of CTB Buer, chloro:isoamyl
alcohol, and BB Buer.
DNA remains bound to
column
Increase elution total volume
to 200 L and incubate
at 65C for 5 min before
centrifugation.
DNA washed o
Dilute DW Buer by adding
appropriate volume of absolute
ethanol prior to use.
Problems in
downstream
applications
Salt carry-over DW Buer must be at room
temperature.
Ethanol carry-over
Following the second wash
spin, ensure that the column is
dried by centrifuging 2 min at
maximum speed.
TROUBLE SHOOTING GUIDE
Please use this guide to troubleshoot any problems that may arise.
For further assistance, please contact the technical support staff, toll free, at
1-800-776-4431.
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Plant DNA Purification Kit
NOTES

Plant DNA Purification Kit
NOTES

935 Cobb Place Blvd
Kennesaw, GA 30144
Phone: [phone]
Fax: [phone]
www.omni-inc.com