E.Z.N.A.® Soil RNA Midi Kit
Table of Contents
Introduction....................................................................2
Illustrated Protocol..........................................................3
Kit Contents/Storage and Stability.................................4
Preparing Reagents.........................................................5
Storage and Quantification of RNA................................6
Soil RNA Midi Protocol....................................................7
Troubleshooting Guide...................................................9
Ordering.........................................................................11
Manual Revision: April 2011
Innovations in nucleic acid isolation
1
Introduction
The E.Z.N.A.® Soil RNA Midi Kit is designed to isolate high quality total RNA from soil
samples typically containing humic acid and inhibitors of RT-PCR. This kit uses a novel
and proprietary method to isolate total RNA from a variety of environmental samples. Soil
samples are homogenized and extracted with phenol/chloroform. A specially formulated
suspension buffer is added to remove coloration and inhibitors. By using an innovative
DNA Clearance Column, DNA is effectively removed without the need for DNase digestion.
The filtrate containing the RNA is then adjusted with ethanol and applied to the HiBind®
RNA Column to which total RNA binds, while cellular debris and other contaminants are
effectively washed away. High quality RNA is finally eluted in DEPC-treated sterile water.
The entire procedure can be completed in under one hour.
This kit has been successfully used to isolated RNA from soil samples collected from forest,
riverbed, grassland, garden and cultivation fields. Isolated RNA can be used for most
downstream applications including RT-PCR, Northern Blot and more.
Binding Capacity
Each HiBind® RNA Midi column can bind approximately 1 mg of RNA.
2
Illustrated Protocol
Homogenize soil sample with lysis buffer, glass
beads, and phenol/chloroform
Transfer Aqueous Phase
Treat with SRE/SRF Suspension
Add Binding Buffer
Pass through DNA Clearance Column
Add EtOH
Bind to HiBind® RNA Column
Wash 3X
Dry
Elute
3
Kit Contents
Product
R6826-00
R6826-01
R6826-02
Number of Purifications
2
10
25
DNA Clearance Midi Column
2
10
25
HiBind® RNA Midi Column
2
10
25
15 mL Collection Tubes
4
20
50
Glass Beads
2.5 g
12 g
30 g
SLX-Mlus Buffer
30 mL
150 mL
375 mL
DS Buffer
0.5 mL
2.5 mL
6 mL
SRE Buffer
20 mL
100 mL
250 mL
SRF Buffer
20 mL
100 mL
250 mL
XP2 Buffer
14 mL
70 mL
175 mL
RWF Buffer
7 mL
35 mL
80 mL
RNA Wash Buffer ll
5 mL
20 mL
50 mL
DEPC-ddH2O
2 mL
10 mL
25 mL
P
P
P
Instruction Manual
Storage and Stability
All components in the Soil RNA Midi Kit should be stored at room temperature. All components are guaranteed for at least 12 months from the date of purchase when stored at
indicated temperature.
4
Preparing Reagents
Dilute RNA Wash Buffer ll with 100% ethanol as follows and store at room temperature:
Kit
Ethanol To Be Added
R6826-00
Add 20 mL absolute ethanol
R6826-01
Add 80 mL absolute ethanol
R6826-02
Add 200 mL absolute ethanol
Prepare water-saturated phenol as follows:
Place the solid phenol in a water bath preset at 75°C until phenol is completely dissolved.
Add an equal volume of molecular biology grade water and mix thoroughly by shaking.
Store the solution at room temperature for 4 hours to overnight until the water phase
(upper phase) and phenol phase (lower phase) are clearly separated. Remove the water
phase with a transfer pipette.
Prepare fresh SRE/SRF Suspension as follows : Important! This suspension must be prepared fresh(1 hour prior to use) for each batch of isolation:
1.
2.
3.
4.
5.
6.
7.
8.
In a 15 mL centrifuge tube, combine 4 mL SRE Buffer and 4 mL SRF Buffer.
Vortex to mix thoroughly. A while precipitation will form. This is normal.
Centrifuge at 4,000 x g for 2 minutes.
Discard the supernatant.
Add 4 mL SLX-Mlus Buffer. Resuspend the pellet by vortexing thoroughly.
Centrifuge at 4,000 x g for 2 minutes.
Discard the supernatant.
Add 2 mL SLX-Mlus Buffer. Resuspend the pellet by vortexing thoroughly.
5
Storage and Quantification of RNA
Storage of RNA
Purified RNA is stable for more than a year when stored at -70°C.
Quantification of RNA
To determine the concentration and purity of RNA, measure absorbance at 260 nm and
280 nm with a spectrophotometer. One OD unit measured at 260 nm corresponds to 40
μg/mL RNA. If it is necessary to dilute RNA sample, use a buffer with neutral pH. The A260/
A280 ratio of pure nucleic acids is 2.0, while an A260/A280 ratio of 0.6 denotes pure protein.
A ratio of 1.8-2.0 corresponds to 90%-100% pure nucleic acid. Phenol has a maximum
absorbance at 270 nm and can interfere with absorbance readings of DNA or RNA.
RNA Quality
It is highly recommended that RNA quality be determined prior to all analysis. The quality
of RNA can be best assessed by denaturing agarose gel electrophoresis and ethidium
bromide staining. Two sharp bands, corresponding to the 28S and 18S (23S and 16S for
bacteria) ribosomal RNA, should be visible on the gel. If these bands appear as smear
towards smaller size, it is likely that RNA has undergone degradation during preparation,
handling, or storage.
6
Soil RNA Midi Protocol
All centrifugation steps used are preformed at room temperature unless otherwise
noted.
Materials and Reagents to be Supplied by User:
•
•
•
•
•
Ethanol (100%)
Water-saturated phenol
Chloroform
Centrifuge with 15 or 50 mL tube adaptor capable of at least 4,000 x g
Sterile RNase-free pipette tips and 15 or 50 mL centrifuge tubes
1.
Add 2 g soil sample, 1 g glass beads and 2 mL SLX-Mlus Buffer to a 15 or 50 mL
centrifuge tube. Vortex for 1 minute to mix thoroughly.
2.
Add 200 μL DS Buffer. Vortex for 30 second to mix thoroughly.
3.
Add 2 mL water-saturated phenol (see Preparing Reagents section) and 2 mL
chloroform. Vortex for 5 minutes to mix thoroughly.
4.
Centrifuge at 4,000 x g for 5 minutes to separate the aqueous and organic phase.
Note: The sample should separate into 3 phases: an upper aqueous phase (often dark
brown) which contains RNA and DNA; a light-colored organic inter phase consists of
phenol/chloroform; and a lower insoluble phase of soil and debris.
5.
Immediately transfer the upper aqueous phase (around 2 mL) to a new 15 mL
centrifuge tube. Take care not to disturb the organic layer.
6.
Add 2 mL freshly-made SRE/SRF Suspension (see Preparing Reagents section). Vortex
to mix thoroughly.
Note: SRE/SRF Suspension must be made fresh for each batch of isolation.
7.
Centrifuge at 4,000 x g for 2 minutes.
8.
Transfer the supernatant to a new 15 mL tube.
9.
Optional: If strong coloration still exists, another 2 mL freshly-made SRE/SRF
Suspension may be added. Repeat Steps 6-8.
10. To the cleared supernatant, add an equal volume of XP2 Buffer. Vortex to mix
thoroughly.
7
Soil RNA Midi Protocol
11. Apply 4 mL of the sample onto the DNA Clearance Midi Column inserted into a 15 mL
collection tube. Centrifuge at 4,000 x g for 5 minutes.
12. Transfer the filtrate to a new 15 mL tube.
13. Repeat Steps 11-12 until all the sample passes through. Combine all the filtrate.
14. Add 0.5 volume of 100% EtOH to the filtrate. Vortex to mix thoroughly.
15. Apply 4 mL of the sample onto the HiBind® RNA Midi Column inserted into a 15 mL
collection tube. Centrifuge at 4,000 x g for 5 minutes.
16. Discard the filtrate. Reinsert the column into the 15 mL tube.
17. Repeat Steps 15-16 until all the sample passes through.
18. Add 3 mL RWF Buffer to the column. Centrifuge at 4,000 x g for 5 minutes. Discard
the filtrate and reuse the collection tube in the next step.
19. Add 4 mL RNA Wash Buffer ll to the Column. Centrifuge at 4,000 x g for 5 minutes.
Discard the filtrate and reuse the collection tube in the next step.
20. Repeat Step 19 one more time.
21. With the column and collection tube empty, centrifuge at 4,000 x g for 15 minutes to
completely dry the HiBind® matrix.
22. Transfer the column to a new 15 mL centrifuge tube and elute the RNA with 500 μL
DEPC-treated water. Make sure to add water directly onto the center of the column
matrix. Centrifuge for 5 minutes at 4,000 x g.
RNA may be eluted with a greater volume of water. While additional elutions
increase total RNA yield, the concentration will be lowered since more than 80% of
RNA is recovered with the first elution. Pre-heating the water to 70°C before adding
to column and incubating the column for 5 minutes at room temperature before
centrifugation may increase yields.
8
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 (800-832-8896).
Possible Problems and Suggestions
Problem
Low RNA yield
Eluted RNA
shows color
Cause
Solution
Incompletely sample
lysis
Increase the vortex speed and duration at
steps 1 and 3. Use a bead beater if available.
Prepare SRE/SRF Suspension according
SRE/SRF Suspension is
to the instruction outlined in Preparing Renot prepared correctly or
agents section. Use only freshly prepared
freshly
SRE/SRF Suspension.
Column is not dried
completely before elution
Make sure the empty column is centrifuged for at least 15 minutes before
elution.
RWB Buffer is not
prepared correctly with
ethanol
Ensure RWB buffer is diluted with 4 volumes of 100% ethanol as indicated on the
bottle.
Prepare SRE/SRF Suspension according
SRE/SRF Suspension is
to the instruction outlined in Preparing Renot prepared correctly or
agents section. Use only freshly prepared
freshly
SRE/SRF Suspension.
Not enough SRE/SRF
Suspension is used
Increase the usage of SRE/SRF Suspension
(Optional Step 9).
Starting sample
Problems
Always use fresh soil sample when possible.
RNase contamination
Ensure not to introduce RNase during the
procedure.
Degraded RNA
Check buffers for RNase contamination.
Problem with
downstream
applications
DNA
contamination
Salt carry-over during
elution
Ensure RWB Buffer is diluted with 4 volumes of 100% ethanol as indicated on the
bottle.
RWB Buffer must be stored and used at
room temperature.
RNA contains inhibitor
from soil
Increase the usage of SRE/SRF Suspension
(Optional Step 9).
Sample has very high
DNA content
Digest with RNase-free DNase and
inactivate DNase by incubate at 65°C for 5
min in the presence of EDTA.
9
Troubleshooting Guide
Problem
Cause
Solution
RNA diluted in acidic
buffer or water
DEPC-treated water is acidic and
can dramatically lower OD260 values.
Use TE buffer to dilute RNA prior to
spectrophotometric analysis.
RNA contains humaic
acids substance
Increase the usage of SRE/SRF Suspension
(Optional Step 9).
Low Abs ratios
10
Ordering Information
The following components are available for purchase separately.
(Call Toll Free at 1-800-832-8896)
Buffer (Size)
RWB Buffer (25 mL)
DEPC Water (100 mL)
Part Number
PDR046
PR032
HiBind®, E.Z.N.A® and MicroElute® are registered trademarks of Omega Bio-tek, Inc.
PCR is a patented process of Hoffman-La Roche. Use of PCR process requires a license.
11
Notes:
12