E-Z 96 Plant DNA DS Kit - Omega Bio-Tek

E-Z 96 Plant DNA DS Kit
D1411-01
D1411-02
1 x 96 preps
4 x 96 preps
August 2015
E-Z 96 Plant DNA DS Kit
Table of Contents
Introduction....................................................................................2
Illustrated Protocols......................................................................3
Kit Contents / Storage and Stability.......................................4
Preparing Reagents / Cleaning Plates....................................5
Guidelines for Vacuum Manifold.............................................6
Disruption of Plant Tissues.........................................................8
E-Z 96 Plant DNA DS Centrifugation Protocol.................10
E-Z 96 Plant DNA DS Vacuum Protocol.............................14
Troubleshooting Guide.............................................................19
Ordering.........................................................................................20
Manual Revision: August 2015
Innovations in nucleic acid isolation
1
Introduction
The E-Z 96 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 E-Z 96 DNA Plate . 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
Centrifugation Protocol
Vacuum Protocol
Collect Plant Tissue
and Homogenize
Collect Plant Tissue
and Homogenize
Lyse
Lyse
Transfer Cleared
Lysate and Adjust
Binding Conditions
Transfer Cleared
Lysate and Adjust
Binding Conditions
Bind and Wash 3X
Bind and Wash 3X
Innovations in nucleic acid isolation
Vacuum
Dry Membrane
Dry Membrane
Innovations in nucleic acid isolation
Elute
Elute
3
Kit Contents
Product Number
D1411-00
D1411-01
E-Z 96 DNA Plate
1
4
1.2 mL HTS Plate
1
4
40 x 8
160 x 8
96-well Square-well Plate (2.2 mL)
2
8
96-well Racked Microtubes
1
4
E-Z 96 Homogenizer Plates
1
4
CSPL Buffer
80 mL
300 mL
Proteinase K Solution
2.2 mL
8.8 mL
XP2 Buffer
60 mL
250 mL
RBB Buffer
60 mL
250 mL
VHB Buffer
44 mL
176 mL
DNA Wash Buffer
40 mL
160 mL
Elution Buffer
15 mL
50 mL
RNase A
550 μL
2.2 mL
P
P
Caps for Racked Microtubes
User Manual
Storage and Stability
All components of the E-Z 96 Plant DNA DS Kit are guaranteed for at least 12 months from
date of purchase when stored as follows. Store RNase A at 2-8° C. All other components
should be stored at room temperature. During shipment or storage in cool ambient
conditions, precipitates may form in VHB Buffer. Dissolve such deposits by warming the
solution at 37°C and gently shaking.
4
Preparing Reagents
•
•
Dilute VHB Buffer with 100% ethanol as follows and store at room temperature.
Kit
100% Ethanol to be Added
D1411-00
56 mL
D1411-01
224 mL
Dilute DNA Wash Buffer with 100% ethanol as follows and store at room temperature.
Kit
100% Ethanol to be Added
D1411-00
160 mL
D1411-01
640 mL
5
Guidelines for Vacuum Manifold
The following is required for use with the Vacuum Protocol:
A) Vacuum Manifold (We recommend Omega Bio-tek’s VAC-03)
Other Compatible Vacuum Manifolds: Qiagen QIAvac24, Sigma AldrichVM20, Promega Vacman®, or manifold with standard Luer connector
B) Vacuum Flask
C) Vacuum Tubing
D) Vacuum Source (review tables below for pressure settings)
Manifold
Recommended Pressure (mbar)
VAC-03
-200 to -400
Conversion from millibars:
Multiply by:
Millimeters of Mercury (mmHg)
0.75
Kilopascals (kPa)
0.1
Inches of Mercury (inchHg)
0.0295
Torrs (Torr)
0.75
Atmospheres (atmos)
0.000987
Pounds per Square Inch (psi)
0.0145
Illustrated Vacuum Setup
Innovations in nucleic acid isolation
Omega Bio-tek’s VAC-03
C) Vacuum Tubing
D) Vacuum Source
A) Vacuum Manifold
B) Vacuum Flask
6
Guidelines for Vacuum Manifold
DNA Bind and Wash Setup
E-Z 96 DNA Plate
Vacuum Manifold Top
Innovations in nucleic acid isolation
Waste Collection
Vacuum Manifold Base
Standard Elution Setup
Optional Elution Setup
E-Z 96 DNA Plate
E-Z 96 DNA Plate
Vacuum Manifold Top
Innovations in nucleic acid isolation
Vacuum Manifold Top
Innovations in nucleic acid isolation
Racked Microtubes
Microplate (300 µL)
Vacuum Manifold
Base
Microplate (300 µL)
Racked Microtubes
Vacuum Manifold
Base
7
Disruption of Plant Tissues
Disrupt Samples With Commercial Homogenizers
A) Dry Specimens
Dried/lyophilized plant tissue can be effectively disrupted and homogenized by rapid
agitation in the presence of beads.
1.
Add one 3-4 mm stainless steel bead to each well of a 1.2 mL HTS plate.
2.
Close the individual tubes with Caps for Racked Microtubes
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.
B) Fresh/Frozen Specimens
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
1.
Add one 3-4 mm stainless steel bead to each well of a 1.2 mL HTS plate.
2.
Close the individual tubes with Caps for Racked Microtubes.
3.
Freeze samples in liquid nitrogen.
Alternative to liquid nitrogen: Sample can be homogenized in presence of 700 µL
CSPL Buffer and 20 µL proteinase K solution for fresh samples. Skip to step 4 of the
E-Z 96 Plant DS Protocol if homogenized in presenece of lysis buffer. Complete steps
4 and 5 of disruption of plant tissue(below):
8
4.
Place the racks or plates into the clamps of the homogenizer.
5.
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 96 Plant DNA DS Kit Protocols
E-Z 96 Plant DNA DS Kit Protocol - Centrifugation Protocol
Materials and Equipment to be Supplied by User:
•
•
•
•
•
•
Centrifuge equipped with swing-bucket rotor capable of at least 3,000 x g
Water baths, ovens, or incubators capable of 65°C
Vortexer
100% ethanol
Liquid nitrogen for freezing/disrupting samples (for fresh/frozen specimens)
Equipment for disrupting plant tissue
Before Starting:
•
•
•
1.
Prepare VHB Buffer, and DNA Wash Buffer according to Preparing Reagents section on
Page 5
Set a water bath, oven, or incubator to 65°C
Heat Elution Buffer to 65°C
Transfer up to 10 mg dry powdered tissue or 50 mg fresh (or frozen) tissue to a 96well Round-well Plate(provided) and seal with Caps for Round-well Plate.
Note: No more than 50 mg (wet weight) or 10 mg (dry weight) starting material is
recommended. More or less can be used depending on results. Water content (and
buffer absorption) of samples affect optimal starting amounts.
2.
Homogenize plant tissue following one of the methods described in the Disruption
of Plant Tissue section on Page 8. If homogenizing in the presence of lysis buffer with
fresh tissue skip to step 4 after homogenization is complete.
3.
Add 700 μL CSPL Buffer and 20 µL Proteinase K Solutionto each sample. Seal the wells
with Caps. Vortex to mix thoroughly.
Note: CSPL Buffer can be mixed with Proteinase K before use. Please see the
Preparing Reagents section on Page 5 for instructions. Ensure that all the samples
are completely suspended and that there are no clumps in the solution. Clumps will
result in low yields.
4.
Incubate at 65°C for 30 minutes. Mix samples twice during incubation by briefly
9
E-Z 96 Plant DNA DS Kit Protocols
shaking the plate side to side.
5.
Centrifuge at 3,000-6,000 x g for 10 minutes.
6.
Remove and discard the caps.
7.
Place the E-Z 96 Homogenizer Plate on to a 96-well Square-well Plate (provided).
8.
Transfer 550 μL cleared supernatant to the E-Z 96 Homogenizer Plate.
9.
Centrifuge at 3,000-6,000 x g for 5 minutes.
10. Add 5 µL Rnase A. Let sit at room temperature for 5 minutes.
11. Add 525 µL RBB Buffer and 525 µL XP2 Buffer. Mix thoroughly by pipetting up and
down or vortexing.
12. Place the E-Z 96 DNA Plate on to a new 96-well Square-well Plate (provided).
13. Carefully transfer 750 µL sample to the E-Z 96 DNA Plate. Be careful not to spill
sample liquid onto the rims of the wells during the transfer.
14. Centrifuge at 3,000-5,000 x g for 5 minutes or until all the sample has passed through
the HiBind® membrane.
15. Discard the filtrate and reuse the 96-well Square-well Plate.
16. Carefully transfer another 750 µL sample to the E-Z 96 DNA Plate. Be careful not to
spill sample liquid onto the rims of the wells during the transfer.
17. Centrifuge at 3,000-5,000 x g for 5 minutes or until all the sample has passed through
the HiBind® membrane.
10
E-Z 96 Plant DNA DS Kit Protocols
18. Discard the filtrate and reuse the 96-well Square-well Plate.
19. Add 500 μL VHB Wash Buffer to each well of the E-Z 96 DNA Plate.
Note: VHB Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 5 for instructions.
20. Centrifuge at 3,000-5,000 x g for 5 minutes.
21. Discard the filtrate and reuse the 96-well Square-well Plate.
22. Add 700 μL DNA Wash Buffer to each well of the E-Z 96 DNA Plate.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 5 for instructions.
23. Centrifuge at 3,000-5,000 x g for 5 minutes.
24. Discard the filtrate and reuse the 96-well Square-well Plate.
25. Repeat Steps 22-24 for a second DNA Wash Buffer wash step.
26. Centrifuge at 3,000-5,000 x g for 15 minutes to dry the plate.
Note: It is important to dry the plate membrane before elution. Residual ethanol may
interfere with downstream applications.
27. Transfer the E-Z 96 DNA Plate to new 96-well Racked Microtubes (provided) or a 96well microplate (not provided).
28. Add 100 μL Elution Buffer heated at 65°C to each well.
29. Incubate at 65°C for 5 minutes.
11
E-Z 96 Plant DNA DS Kit Protocols
30. Centrifuge at 5,000 x g for 5 minutes.
31. Repeat Steps 28-30for a second elution step.
Note: To maintain higher DNA concentration, second elution may be performed
with first eluate.
32. Seal the 96-well Racked Microtubes with Caps for Racked Microtubes.
33. Store DNA at -20°C.
12
E-Z 96 Plant DNA DS Kit Protocols
E-Z 96 Plant DNA DS Kit - Vacuum Protocol
The following protocol is based on using Omega Bio-tek’s vacuum manifold (Cat# VAC-03).
Materials and Equipment to be Supplied by User:
•
•
•
•
•
•
•
•
•
Vacuum manifold and vacuum source
Centrifuge equipped with swing-bucket rotor capable of at least 3,000 x g
Water bath, oven, or incubator capable of 80°C
Vortexer
100% ethanol
Liquid nitrogen for freezing/disrupting samples (For Fresh/Frozen Specimens)
Equipment for disrupting plant tissue
Ice, freezer, or 96-well cryorack at -20°C
Sealing film
Before Starting:
•
•
•
1.
Prepare HBC Buffer and DNA Wash Buffer according to Preparing Reagents section on
Page 5
Set a water bath, oven, or incubator to 65°C
Heat Elution Buffer to 65°C
Transfer up to 10 mg dry powdered tissue or 50 mg fresh (or frozen) tissue to a 96well Round-well Plate(provided) and seal with Caps for Round-well Plate.
Note: No more than 50 mg (wet weight) or 10 mg (dry weight) starting material is
recommended. More or less can be used depending on results. Water content (and
buffer absorption) of samples affect optimal starting amounts.
2.
Homogenize plant tissue following one of the methods described in the Disruption
of Plant Tissue section on Pages 8. If homogenizing in the presence of lysis buffer
with fresh tissue skip to step 4 after homogenization is complete.
3.
Add 700 μL CSPL Buffer and 20 µL Proteinase K Solutionto each sample. Seal the wells
with Caps. Vortex to mix thoroughly.
Note: CSPL Buffer can be mixed with Proteinase K before use. Please see the
Preparing Reagents section on Page 5 for instructions. Ensure that all the samples
are completely suspended and that there are no clumps in the solution. Clumps will
result in low yields.
13
E-Z 96 Plant DNA DS Kit Protocols
4.
Incubate at 65°C for 30 minutes. Mix samples twice during incubation by briefly
shaking the plate side to side.
5.
Centrifuge at 3,000-6,000 x g for 10 minutes.
6.
Remove and discard the caps.
7.
Place the E-Z 96 Homogenizer Plate on to a 96-well Square-well Plate (provided).
8.
Transfer 550 μL cleared supernatant to the E-Z 96 Homogenizer Plate.
9.
Centrifuge at 3,000-6,000 x g for 5 minutes.
10. Add 5 µL Rnase A. Let sit at room temperature for 5 minutes.
11. Add 525 µL RBB Buffer and 525 µL XP2 Buffer. Mix thoroughly by pipetting up and
down or vortexing.
12. Prepare the vacuum manifold according to manufacturer’s instructions.
13. Place an E-Z 96 DNA Plate on the top part of the vacuum manifold. Place the waste
collection tray inside the base of the manifold. Seal any unused wells with sealing
film (not provided).
14. Transfer 750 µL sample to the E-Z 96 DNA Plate.
15. Turn on the vacuum source to draw the sample through the plate.
16. Turn off the vacuum.
17. Repeat Steps 14-16 until all the sample has been transferred to the E-Z 96 DNA Plate.
14
E-Z 96 Plant DNA DS Kit Protocols
18. Add 500 µL VHB Wash Buffer to each well.
Note: VHB Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 5 for instructions.
19. Turn on the vacuum source to draw the VHB Wash Buffer through the plate.
20. Turn off the vacuum.
21. Add 700 µL DNA Wash Buffer to each well.
Note: DNA Wash Buffer must be diluted with 100% ethanol prior to use. Please see
Page 5 for instructions.
22. Turn on the vacuum source to draw the DNA Wash Buffer through the plate.
23. Turn off the vacuum.
24. Repeat Steps 21-23 for a second DNA Wash Buffer wash step.
25. Add 400 µL 100% ethanol to each well.
26. Turn on the vacuum source to draw the ethanol through the plate.
27. Turn off the vacuum.
28. Continue to apply the vacuum for 10 minutes after all liquid has passed through the
E-Z 96 DNA Plate.
29. Turn off the vacuum.
30. Discard and remove the filtrate and waste collection plate.
15
E-Z 96 Plant DNA DS Kit Protocols
31. Place the E-Z 96 DNA Plate upside down on a stack of paper towels and tap several
times to remove any residual ethanol.
Note: It is very important to completely dry the E-Z 96 DNA Plate before elution. If a
swing bucket centrifuge with a 96-well plate adaptor is available, centrifuge at 5,000
x g for 5 minutes to dry the plate. Or if an oven/incubator is available, dry the plate at
65°C for 10 minutes.
32. Place the 96-well Racked Microtubes inside the base of the manifold.
33. Place the E-Z 96 DNA Plate on top of the manifold.
34. Add 100 µL Elution Buffer heated to 65°C to each well.
35. Let sit at room temperature for 5 minutes.
36. Turn on the vacuum source to draw the Elution Buffer through the plate.
37. Turn off the vacuum.
Optional: Repeat Steps 34-37 for a second elution step.
Note: 100 µL Elution Buffer is sufficient to elute up to 85% of the DNA from each well
of the E-Z 96 DNA Plate. A second elution step with same 100 µL elute containing
DNA, reheated to 65°C, will increase yield by up to 10-15%. Total DNA yields vary
depending on type and quantity of sample.
38. Seal the 96-well Racked Microtubes with Caps for Racked Microtubes (provided).
39. Store DNA at -20°C.
16
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.
Possible Problems and Suggestions
Problem
Cause
Solution
Clogged well
Sample too viscous
Do not exceed suggested amount of
starting material.
Problem
Cause
Solution
Incomplete disruption of
Completely homogenize sample.
starting material
Low DNA yield
Poor lysis of tissue
Decrease the amount of starting material
or increase the amount of CSPL Buffer
DNA remains bound to
column
Increase elution volume to 200 µL and
incubate the plate at 65°C for 5 minutes
before centrifugation.
DNA washed off
Dilute SPW Wash Buffer by adding
appropriate volume of 100% ethanol
prior to use (Page 5).
If 550 µL lysis buffer cannot be
transferred after clearing lysate by
Insufficient sample
centrifugation. Increase volume of CSPL
amount transferred afBuffer. If only 350 µL could be recovered
ter supernatant removal then increase amount by 200 µL(550
µL Desired amount- 350 µL= 200 µL
additional lysis buffer amount required.
Problem
Problems in
downstream
applications
Cause
Solution
Salt carryover
Repeat wash step with SPW Wash Buffer.
Ethanol carryover
Following the second wash spin, ensure
that the plate is completely dried before
elution.
17
Ordering Information
The following components are available for purchase separately.
(Call Toll free at 1-800-832-8896)
Product
Part Number
Elution Buffer (100 mL)
PDR048
Sealing Film
AC1200
AeraSeal Film
AC1201
96-well Square-well Plate (2.2 mL)
EZ9602
E-Z 96 DNA Plates (10)
BD96-01
E-Z 96 Homogenizer Plates (4 x 96)
HCR9601-02
E-Z 96 Lysate Clearance Plates (10 x 96)
FL9601
Vacuum Manifold
VAC-03
HiBind®, E.Z.N.A.®, and MicroElute® are registered trademarks of Omega Bio-tek, Inc.
Qiagen®, QIAvac® and Vacman® are all trademarks of their respected companies.
PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license.
18
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