E.Z.N.A.® Plant DNA DS Mini Kit
D2411-00
D2411-01
May 2015
5 preps
50 preps
E.Z.N.A.® Plant DNA DS Mini Kit
Table of Contents
Introduction and Overview.......................................................2
Kit Contents/Storage and Stability.........................................3
Preparing Reagents......................................................................4
Disruption/Homogenization.....................................................5
Protocol for Fresh/Frozen/Dry Samples................................7
Troubleshooting Guide.............................................................10
Ordering.........................................................................................11
Manual Revision: May 2015
Innovations in nucleic acid isolation
1
Introduction and Overview
The E.Z.N.A.® Plant DNA DS Mini Kit is designed for efficient recovery of genomic DNA up
to 30 kb in size from fresh, frozen, or dried plant tissue samples rich in polysaccharides,
polyphenols, or those having a lower DNA content. Up to 50 mg wet tissue can be
processed in less than 1 hour. The system combines the reversible nucleic acid-binding
properties of the HiBind® 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.
This procedure relies on the well established properties of the cationic detergent,
cetyltrimethyl ammonium bromide (CTAB), in conjunction with the unique binding
system to increase yields and provide high-quality DNA. The system eliminates the
need for chloroform extractions traditionally associated with CTAB-based lysis methods.
Samples are homogenized and lysed in a high salt buffer containing CTAB, binding
conditions are adjusted, and DNA is purified using a HiBind® 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.
2
Kit Contents
Product
D2411-00
D2411-01
Purifications
5
50
HiBind® DNA Mini Columns
5
50
Homogenizer Mini Columns
5
50
2 mL Collection Tubes
10
100
5 mL
20 mL
150 µL
1.5 mL
RBB Buffer
5 mL
30 mL
XP2 Buffer
5 mL
30 mL
RNase A (25 mg/mL)
30 µL
300 µL
HBC Buffer
4 mL
25 mL
DNA Wash Buffer
1.5 mL
25 mL
Elution Buffer*
1.5 mL
15 mL
P
P
CSPL Buffer
Proteinase K Solution
User Manual
* 10 mM Tris HCl pH 8.5
Storage and Stability
All of the E.Z.N.A.® Plant DNA DS Mini Kit components are guaranteed for at least 12
months from the date of purchase when stored as follows. RNase A must be stored at
2-8°C. All remaining components should be stored at room temperature. During shipment
or storage in cool ambient conditions, precipitates may form in HBC Buffer. Dissolve such
deposits by warming the solution at 37°C and gently shaking.
3
Preparing Reagents
1.
2.
4
Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.
Kit
100% Ethanol to be Added
D2411-00
6 mL
D2411-01
100 mL
Dilute HBC Buffer with 100% isopropanol as follows and store at room temperature.
Kit
100% Isopropanol to be Added
D2411-00
1.6 mL
D2411-01
10 mL
Disruption of Plant Tissues
1.
Grind samples with pestle
A) Dry Specimens
Drying allows storage of field specimens for prolonged periods of time prior
to processing. Samples can be dried overnight in a 45°C oven, powdered, and
stored dry at room temperature. To prepare dried samples, place ~15 mg of
dried tissues into a microcentrifuge tube (1.5 mL tubes are recommended) and
grind using a pellet pestle. Disposable Kontes pestles work well and are available
from Omega Bio-tek (Cat# SSI-1014-39 & SSI-1015-39). For critical work such
as PCR and cloning, pestles are best used a single time then soaked in a dilute
bleach solution immediately after use until clean. Disposable pestles may be
autoclaved several times. A fine powder will ensure optimal DNA extraction and
yield.
B) Fresh/Frozen Specimens
Due to the tremendous variation in water and polysaccharide content of plants,
sample size should be limited to ~30 mg for first time users. It is very important
to not overload the HiBind® DNA Mini Column. Too much starting material will
decrease the yield and purity due to inefficient lysis. However, for some plant
species, increasing the starting material can increase DNA yield. We recommend
starting with 30 mg tissue. If results obtained are satisfactory, then increase
amount of starting material. Best results are obtained with young leaves or
needles.
To prepare samples, collect tissue in a 1.5 mL or 2 mL microcentrifuge tube and
dip the tube in liquid nitrogen with a pair of tweezers to fill the tube. Grind
the tissue using disposable Kontes pellet pestles, which are available from
Omega Bio-tek (Cat# SSI-1015-39). Alternatively, allow the liquid nitrogen to
evaporate and store the samples at -70°C for later use. For critical work such
as PCR and cloning, pestles are best used a single time then soaked in a dilute
bleach solution immediately after use until clean. Disposable pestles may be
autoclaved several times. For standard Southern analysis, the same pestle can
be reused several times to grind multiple tissue samples by rinsing with ethanol
and carefully wiping the surfaces clean between samples. Transfer the ground
sample into a 1.5 mL microcentrifuge tube.
Note: Do not allow the sample to thaw during handling and weighing. To
prevent the sample from thawing, keep the samples on a bed of dry ice.
5
Disruption of Plant Tissues
1.
Disrupt Samples With Commercial Homogenizers
Fresh, frozen, and dried plant tissue can be effectively disrupted and homogenized
by rapid agitation in the presence of beads.
For Fresh, Frozen and Lyophilized/Dried Tissue
6
1.
Add two 3-4 mm stainless steel bead or ceramic beads to each vial.
2.
Close the individual vial.
3.
Place the racks or plates into the clamps of the homogenizer.
4.
Homogenize for 60-90 seconds at 30 Hz. Tissue samples are disrupted and
simultaneously homogenized with the shearing and crushing action of the
beads.
E.Z.N.A.® Plant DNA DS Mini Kit
E.Z.N.A.® Plant DNA DS Mini Kit - Wet/Frozen/Dry Tissue Protocol
Materials and Equipment to be Supplied by User:
•
•
•
•
•
•
•
Microcentrifuge capable of at least 12,000 x g
Waterbath capable of 65°C
Vortexer
Nuclease-free 1.5 and 2 mL microcentrifuge tubes
100% isopropanol
100% ethanol
Sample Disruption Method (See Pages 5-6)
Before Starting:
•
•
Prepare the DNA Wash Buffer and HBC Buffer according to the instructions on Page 4.
Heat the Elution Buffer to 65°C.
1.
Prepare 10-50 mg wet/frozen tissue or 2-10 mg dry tissue in a 1.5 or 2 mL
microcentrifuge tube/vial (not provided) according to Pages 5-6. For best results use
a commercial homogenizer if available.
2.
Add 700 μL CSPL Buffer and 20 µL Proteinase K Solution. Vortex vigorously to mix.
Make sure to disperse all clumps.
3.
Incubate at 65°C for 30 minutes.
4. Centrifuge at 12,000 x g for 3 minutes.
5.
Insert a Homogenizer Mini Column into a 2 mL Collection Tube.
6.
Transfer 550 μL cleared supernatant to the Homogenizer Mini Column.
7.
Centrifuge at 12,000 x g for 1 minute.
7
E.Z.N.A.® Plant DNA DS Mini Kit Protocol
8.
9.
Transfer the filtrate to a new 2 mL microcentrifuge tube (not provided).
Add 5 µL RNase A. Let sit at room temperature for 5 minutes.
10. Add 525 µL RBB Buffer and 525 µL XP2 Buffer. Vortex to mix thoroughly.
11. Insert a HiBind® DNA Mini Column into a 2 mL Collection Tube.
12. Transfer 750 µL lysate from Step 9 to the HiBind® DNA Mini Column.
13. Centrifuge at 12,000 x g for 1 minute.
14. Discard the filtrate and reuse the collection tube.
15. Repeat Steps 12-14 to transfer the remaining lysate.
16. Add 500 µL HBC Buffer.
Note: HBC Buffer must be diluted with isopropanol before use. Please see Page 4 for
instructions.
17. Centrifuge at 12,000 x g for 1 minute.
18. Discard the filtrate and reuse collection tube.
19. Add 700 µL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 4 for instructions.
20. Centrifuge at 12,000 x g for 1 minute.
21. Discard the filtrate and reuse the collection tube.
8
E.Z.N.A.® Plant DNA DS Mini Kit Protocol
22. Repeat Steps 19-21 for a second DNA Wash Buffer wash step.
23. Centrifuge the empty HiBind® DNA Mini Column for 2 minutes at 12,000 x g to dry
the column matrix.
Note: It is important to dry the HiBind® DNA Mini Column matrix before elution.
Residual ethanol may interfere with downstream applications.
24. Transfer the HiBind® DNA Mini Column to a clean 1.5 mL microcentrifuge tube (not
provided).
25. Add 50-100 μL Elution Buffer heated to 65°C directly to the center of the column
membrane.
26. Let sit at room temperature for 1 minute.
27. Centrifuge at 12,000 x g for 1 minute.
28. Transfer the filtrate to the center of the HiBind® DNA Mini Column membrane.
29. Let sit at room temperature for 1 minute.
30. Centrifuge at 12,000 x g for 1 minute.
31. Store filtrate containing DNA at -20°C.
9
Troubleshooting Guide
Please use this guide to troubleshoot any problems that may arise. For further assistance,
please contact the technical support staff, toll free, at 1-800-832-8896.
Problem
Cause
Solution
Carryover of debris
Following transfer from Step 7, cell debris
may have transferred.
Sample too viscous
Do not exceed suggested amount of
starting material.
Cause
Solution
Incomplete disruption
of starting material
For both dry and fresh samples, obtain a fine
homogeneous powder before adding CSPL
Buffer.
Poor lysis of sample
Decrease amount of starting material
or increase amount of CSPL Buffer and
Proteinase K Solution.
DNA remains bound to
column
Increase elution volume to 200 μL and incubate on column at 65°C for 5 minutes before
centrifugation.
DNA washed off
Dilute DNA Wash Buffer by adding
appropriate volume of 100% ethanol prior
to use.
Difficulty transferring
sample after lysis
If you are unable to transfer 550 µL after
lysis with CSPL Buffer increase the buffer
amount so that 550 µL can be successfully
transferred.
Cause
Solution
Salt carryover
DNA Wash Buffer must be at room
temperature.
Ethanol carryover
Following the second wash spin, ensure
that the column is dried by centrifuging 2
minutes at maximum speed.
Clogged
column
Problem
Low DNA
yield
Problem
Problems in
downstream
applications
10
Ordering Information
The following components are available for purchase separately.
(Call Toll Free at 1-800-832-8896)
Product
Part Number
DNase/RNase-free microcentrifuge tubes, 1.5 mL, 500/pk, 10 pk/cs
SSI-1210-00
DNase/RNase-free microcentrifuge tubes, 2.0 mL, 500/pk, 10 pk/cs
SSI-1310-00
HiBind® DNA Mini Columns (200)
DNACOL-02
Homogenizer Mini Columns (200)
HCR003
Elution Buffer (100 mL)
PDR048
DNA Wash Buffer (100 mL)
PS010
RNase A (400 µL)
AC117
HiBind®, E.Z.N.A.®, and MicroElute® are registered trademarks of Omega Bio-tek, Inc.
Qiagen®, QIAvac® and Vacman® are all trademarks of their respective companies.
PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license.
11
Notes:
12