Download FavorPrep Plant Total RNA Mini Kit

FavorPrep Plant Total
RNA Mini Kit
TM
FavorPrep Plant Total RNA Mini Kit
Cat. No.: FAPRK 000
FAPRK 001
FAPRK 001-1
FAPRK 001-2
(For Research Use Only)
Kit Contents:
Cat. No:
FAPRK 000-Mini
(4 preps_sample)
FARB Buffer
FAPRB Buffer
Wash Buffer 1
a
Wash Buffer 2 (concentrate)
RNase-free Water
Filter Column
FARB Mini Column
Collection Tube
Elution Tube
User Manual
3 ml
3 ml
3 ml
1.5 ml
0.5 ml
4 pcs
4 pcs
8 pcs
4 pcs
1
FAPRK 001
(50 preps)
FAPRK 001-1
(100 preps)
FAPRK 001-2
(300 preps)
30 ml
30 ml
30 ml
20 ml
6 ml
50 pcs
50 pcs
100 pcs
50 pcs
1
60 ml
60 ml
60 ml
35 ml
6 ml
100 pcs
100 pcs
200 pcs
100 pcs
1
170 ml
170 ml
170 ml
50 ml x 2
8 ml x 2
300 pcs
300 pcs
600 pcs
300 pcs
1
140 ml
200 ml
Preparation of Wash Buffer by adding ethanol (96 ~ 100%)
a
Ethanol volume for Wash Buffer
6 ml
80 ml
Specification:
Principle: mini spin column (silica matrix)
7
Sample size: up to 100 mg plant tissue or 1 x10 plant cells
Operation time: 30 ~ 60 minutes
Binding capacity: up to 100 µg total RNA/ column
Expected yield: 5 ~30 µg of total RNA from 100 mg of young leave
Column applicability: centrifugation and vaccum
Minimum elution volume: 30 µl
Important Notes:
1. Make sure everything is RNase-free when handling RNA.
2. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers.
3. Pipet a required volume of FARB Buffer or FAPRB Buffer to another RNase-free container and add 10 μl ß-mercaptoethanol
(ß-ME) per 1ml FARB Buffer or FAPRB Buffer before use. Caution: ß-mercaptoethanol is hazardous to human health. perform
the procedures involving FARB Buffer or FAPRB Buffer in a chemical fume hood.
4. Add required volume of RNase-free ethanol (96~100%) to Wash Buffer 2 when first use.
5. All centrifuge steps are done at full speed (~18,000 x g) in a microcentrifuge.
6. Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10 mM MnCl2, 20 mM Tris-HCl, pH 7.0 at 25ºC) to final conc. = 0.5 U/μl.
Brief procedure:
Grind plant sample in liquid nitrogen
Lysis (FARB Buffer)
or (FAPRB Buffer)
centrifuge
Filtration
Add 70% ethanol
centrifuge
centrifuge
RNA Binding
(FARB Mini Column)
optional step: DNase digestion
centrifuge
Washing
(Wash Buffer 1)
(Wash Buffer 2, twice)
On-Column DNase I Digesion
(DNase I / reaction buffer),
15 min
centrifuge
centrifuge
RNA Elution
(RNase-free water)
Washing
(Wash Buffer 1)
Washing
(Wash Buffer 1)
(Wash Buffer 2 x 2)
1
v 0415
General Protocol:
Please Read Important Notes Before Starting Following Steps.
1. Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and transfer to a new microcentrifuge tube (not
provided).
-- Note: Do not use plant sample more than 100 mg, it will lower the total RNA yield.
2. Add 500 µl of FARB Buffer (ß-ME added) to the sample powder and vortex vigorously. Incubate at room temperature for 5 min.
Use FAPRB Buffer (ß-ME added) if plant sample contains sticky secondary metabolites such as maize with milky endosperm or
mycelia of filamentous fungi.
-- Note: In order to release all the RNA from sample, it is required to disrupt the sample completely by using a suitable disruptor
equipment.
3. Place a Filter Column to a Collection Tube and transfer the sample mixture to the Filter Column. Centrifuge at full speed
(~ 18,000 x g) for 1 min.
4. Transfer the clarified supernatant from the Collection Tube to a new microcentrifuge tube (not provided), and adjust the
volume of the supernatant.
-- Note: Avoid to pipette any debris and pellet when transfering the supernatant.
5. Add 1 volume of 70 % RNase-free ethanol and mix well by vortexing.
6. Place a FARB Mini Column to a Collection Tube and transfer the ethanol added sample mixture (including any precipitate) to
the FARB Mini Column. Centrifuge at full speed for 1 min, discard the flow-through and return the FARB Mini Column back to
the Collection Tube.
7. Repeat step 6 for the rest of the sample mixture.
8. (Optional): To eliminate genomic DNA contamination, follow the steps from 8a. Otherwise, proceed to step 9 directly.
8a. Add 250 µl of Wash Buffer 1 to the FARB Mini Column, centrifuge at full speed for1 min. Discard the flow-through
and return the FARB Mini Column back to the Collection Tube.
8b. Add 60 µl of RNase-free DNase 1 solution (0.5U/ul, not provided) to the membrane center of FARB Mini Column.
Place the column on the benchtop for 15 min.
8c. Add 250 µl of Wash Buffer 1 to the FARB Mini Column, centrifuge at ful speed for 1 min. Discard the flow-through
and return the FARB Mini Column back to the Collection Tube.
8d. After DNase 1 treatment, proceed to step 10.
9. Add 500 µl of Wash Buffer 1 to the FARB Mini Column, centrifuge at full speed for 1 min. Discard the flow-through and return
the FARB Mini Column back to the Collection Tube.
10. Add 750 µl of Wash Buffer 2 to the FARB Mini Column, centrifuge at full speed for 1 min. Discard the flow-through and return
the FARB Mini Column back to the Collection Tube.
-- Note: Make sure that ethanol has been added into Wash Buffer 2 when first use.
11. Repeat step 10 for one more washing.
12. Centrifuge the FARB Mini Column at full speed for an additional 3 min to dry the FARB Mini Column.
-- Important Step! This step will avoid the residual liquid to inhibit subsequent enzymatic reaction.
13. Place the FARB Mini Column to a Elution Tube (provided, 1.5 ml microcentrifuge tube).
14. Add 30 ~ 50 µl of RNase-free ddH2O to the membrane center of the FARB Mini Column. Stand the FARB Mini Column for 1 min.
-- Important Step! For effective elution, make sure that RNase-free ddH2O is dispensed on the membrane center and is
absorbed completely.
-- Important : Do not elute the RNA using RNase-free water less than suggested volume (< 30 µl). It will lower the final yield.
15. Centrifuge the FARB Mini Column at full speed for 1 min to elute RNA.
16. Store RNA at -70C.
2
FavorPrep Plant Total
RNA Maxi Kit
TM
FavorPrep Plant Total RNA Purification Maxi
(Cat.: FAPRK 000-Maxi, 2 Preps)
Sample Kit
(For Research Use Only) v.1012
Kit Contents
FAPRK 000-Maxi
(2 Preps)
FARB Buffer
12 ml
FAPRB Buffer
Wash Buffer 1
12 ml
10 ml
Wash Buffer 2* (concentrated)
5 ml
RNase-free water
1 ml
Filter Column
2 pcs
FARB Maxi Column
2 pcs
Brief Procedure
Homogenized plant sample
under liquid nitrogen
Lysis (TRX Buffeer)
70 °C for 10 min
centrifuge
Filtration
centrifuge
Total RNA Binding
centrifuge
Washing (Wash1)
(Wash2)
centrifuge
Total RNA Elution
* Add 20 ml of RNase-free ethanol (96-100 %) to Wash Buffer 2
when first open.
Specifications
7
Sample Amount: 500 mg (up to 1 g) plant tissue or 5~10 X 10 plant
cells
Operation time: about 45~60 min
Binding Capacity: up to 1000 µg total RNA
Expected Yield: up to 50~300 µg total RNA from young leave
(RNase-free Water)
Elution volume: 500 µl
Important Notes
1. Make sure everything is RNase-free when handling RNA.
2. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers.
3. Pipet 5 ml of FARB Buffer to another RNase-free container and add 50 µl of ß-mercaptoethanol (ß-ME) before
every preparation.
4. Add required amount of ethanol (96-100%) Wash Buffer 2 when first open.
5. Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10mM MnCl2, 20mM Tris-HCl, pH7.0 at 25 °C) to final
conc.= 0.5U/µl.
General Protocol:
Please Read Important Notes Before Starting The Following Steps.
1. Grind 500 mg (up to 1 g) of plant sample under liquid nitrogen to a fine powder and transfer to a new 50 ml
centrifuge tube (not provided).
--Note: Do not use plant sample more than 1g, it will lower the total RNA yield.
2. Add 5 ml of FARB Buffer (ß-ME added) to the sample powder and vortex vigorously. Use FAPRB Buffer
(ß-ME added) if plant sample contains sticky secondary metabolites such as maize with milky endosperm
or mycelia of filamentous fungi.
Note: In order to release all the RNA in the sample, it is required to disrupt the sample completely.
Different samples require different methods (ex: disruptor equipment) to achieve complete disruption.
1
3. Incubate at 70 °C for 10 min, vortex every 3 min during incubation.
4. Place a Filter Column to a 50 ml centrifuge tube (not provided). And transfer the entire sample mixture to the
Filter Column.
5. Centrifuge at full speed (4500~6,000 rpm) for 5 min at 4 °C.
6. Transfer the clarified flow-through to a new 50 ml centrifuge tube (not provided) and adjust the volume of the
clarified flow-through.
--Avoid pipett any debris and pellet when transfering the clarified flow-through.
7. Add 1 volume of 70 % ethanol to the clarified flow-through and mix well by plus-vortexing for 5 seconds.
--For example, add 4.5 ml of 70 % ethanol to 4.5 ml of clearified flow-through.
8. Place a FARB Maxi Column to a 50 ml centrifuge tube (not provided). Transfer the ethanol added sample
mixture (including any precipitate) to the FARB Maxi Column. Centrifuge at full speed (4500~6,000 rpm)
for 1 min. Discard the flow-through and place the FARB Maxi Column back to the 50 ml centrifuge tube.
9.(Optional): To eliminate genomic DNA contamination of RNA, follow the steps from 9a. Otherwise, proceed
to step 10 directly.
9a. Add 2.5 ml of Wash Buffer 1 to the FARB Maxi Column. Centrifuge at full speed (4500~6,000 rpm) for 2 min.
Discard the flow-through and place the FARB Maxi Column back to the 50 ml centrifuge tube.
9b. Add 800 µl of RNase-free DNase 1 solution (0.5 U/µl, not provided) to the membrane center of FARB Maxi
Column. Place the Column on the benchtop for 15 min.
9c. Add 2.5 ml of Wash Buffer 1 to the FARB Maxi Column. Centrifuge at full speed (4500~6,000 rpm) for 2 min.
Discard the flow-through and place the FARB Maxi Column back to the 50 ml centrifuge tube.
9d. After DNase 1 treatment, proceed to step 11.
10. Add 5 ml of Wash Buffer to 1 wash the FARB Maxi Column, Centrifuge at full speed (4500~6,000 rpm) for 2 min.
Discard the flow-through and place the FARB Maxi Column back to the 50 ml centrifuge tube.
11. Wash FARB Maxi Column twice with 5 ml of Wash Buffer 2 by Centrifuge at full speed (4500~6,000 rpm) for
2 min. Discard the flow-through and place the FARB Maxi Column back to the 50 ml centrifuge tube.
--Make sure that ethanol has been added into Wash 2 Buffer when first open.
12. Centrifuge at full speed (4500~6,000 rpm) for an additional 10 min to dry the FARB Maxi column.
--Important Step! This step will avoid the residual liquid to inhibit subsequent enzymatic reaction.
13. Place the FARB Maxi Column to a new 50 ml centrifuge (not provided).
14. Add 1 ml of RNase-free Water to the membrane center of the FARB Maxi Column. Stand the FARB Maxi
Column for 5 min.
--Important Step! For effective elution, make sure that the elution solution is dispensed of the membrane
center and is absorbed completely.
15. Centrifuge at full speed (4500~6,000 rpm) for 5 min to elute RNA.
16. Store RNA at -70 °C.
2
FavorPrep
Plant Total RNA Maxi kit
TM
User Manual
Cat. No.: FAPRK 002 (10 Preps)
FAPRK 002-1 (24 Preps)
For Research Use Only
v.1310
Introduction
FavorPrep™ Plant Total RNA Maxi Kit provides a fast and simple method
to isolate total RNA from plant tissue and cells. In the process, sample is
homogenized by grinding the plant tissue in liquid nitrogen and filtrated
by filter column to remove cell debris. In the presence of binding buffer
with chaotropic salt, the total RNA in the lysate binds to glass fiber matrix
in the spin column. the optional DNase treatments can remove DNA
residues and the contaminants are washed with an ethanol contained
wash buffer. Finally, the purified total RNA is eluted by RNase-free water.
The protocol does not require phenol extraction and alcohol precipitation. The entire procedure can be completed in 60 minutes. The purified
total RNA is ready for RT, RT-PCR, real-time PCR, Northern blotting.
13. Place the FARB Maxi Column to a new 50 ml centrifuge (not provided).
14. Add 1 ml of RNase-free Water to the membrane center of the FARB
Maxi Column. Stand the FARB Maxi Column for 5 min.
--Important Step! For effective elution, make sure that the elution solution
is dispensed of the membrane center and is absorbed completely.
15. Centrifuge at full speed (4500~6,000 rpm) for 5 min to elute RNA.
16. Store RNA at -70 °C.
Sample amount and yield:
7
Sample Amount: 500 mg (up to 1 g) plant tissue or 5~10 X 10 plant cells
Operation time: about 45~60 min
Binding Capacity: up to 1000 µg total RNA
Expected Yield: up to 50~300 µg total RNA from young leave
Elution volume: 500 µl
Kit Contents
Cat. No.
/ preps
FAPRK 002
(10 preps)
FAPRK 002-1
(24 preps)
FARB Buffer
60 ml
140 ml
FAPRB Buffer
60 ml
140 ml
60 ml
140 ml
Wash Buffer 1
Wash Buffer 2 (concentrated)
12.5 ml *
RNase-free Water
50 ml **
6 ml
30 ml
Filter Column
10 pcs
24 pcs
FARB Maxi Column
10 pcs
24 pcs
* Add 50 ml ethanol (96-100 %) to Wash Buffer 2 when first open.
** Add 200 ml ethanol (96-100 %) to Wash Buffer 2 when first open.
1
6
Important notes
8. Place a FARB Maxi Column to a 50 ml centrifuge tube (not provided).
Transfer the ethanol added sample mixture (including any precipitate)
to the FARB Maxi Column. Centrifuge at full speed (4500~6,000 rpm)
for 1 min. Discard the flow-through and place the FARB Maxi Column
back to the 50 ml centrifuge tube.
9.(Optional): To eliminate genomic DNA contamination of RNA, follow
the steps from 9a. Otherwise, proceed to step 10 directly.
9a. Add 2.5 ml of Wash 1 Buffer to the FARB Maxi Column. Centrifuge
at full speed (4500~6,000 rpm) for 2 min. Discard the flow-through
and place the FARB Maxi Column back to the 50 ml centrifuge tube.
1. Make sure everything is RNase-free when handling RNA.
2. Buffers provided in this system contain irritants. Wear gloves and lab
coat when handling these buffers.
3. Pipet 5 ml of FARB Buffer to another RNase-free container and add 50 µl of
ß-mercaptoethanol (ß-ME) before every preparation.
4. Add required amount of ethanol (96-100%) Wash 2 Buffer when first open.
5. Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10mM MnCl2,
20mM Tris-HCl, pH7.0 at 25 °C) to final conc.= 0.5U/µl.
9b. Add 800 µl of RNase-free DNase 1 solution (0.5 U/µl, not provided)
to the membrane center of FARB Maxi Column. Place the Column
on the benchtop for 15 min.
9c. Add 2.5 ml of Wash 1 Buffer to the FARB Maxi Column. Centrifuge
at full speed (4500~6,000 rpm) for 2 min. Discard the flow-through
and place the FARB Maxi Column back to the 50 ml centrifuge tube.
9d. After DNase 1 treatment, proceed to step 11.
10. Add 5 ml of Wash 1 Buffer to wash the FARB Maxi Column, Centrifuge
at full speed (4500~6,000 rpm) for 2 min. Discard the flow-through
and place the FARB Maxi Column back to the 50 ml centrifuge tube.
11. Wash FARB Maxi Column twice with 5 ml of Wash 2 Buffer by Centrifuge
at full speed (4500~6,000 rpm) for 2 min. Discard the flow-through
and place the FARB Maxi Column back to the 50 ml centrifuge tube.
--Make sure that ethanol has been added into Wash 2 Buffer
when first open.
12. Centrifuge at full speed (4500~6,000 rpm) for an additional 10 min to
dry the FARB Maxi column.
--Important Step! This step will avoid the residual liquid to inhibit subsequent
enzymatic reaction.
5
2
Brief Procedure
Genernal Protocol:
Please Read Important Notes Before Starting The Following Steps.
Homogenized plant sample
under liquid nitrogen
Lysis (TRX Buffeer)
70 °C for 10 min
1. Grind 500 mg (up to 1 g) of plant sample under liquid nitrogen to a fine
powder and transfer to a new 50 ml centrifuge tube (not provided).
--Note: Do not use plant sample more than 1g, it will lower
the total RNA yield.
2. Add 5 ml of FARB Buffer (ß-ME added) to the sample powder and
vortex vigorously. Use FAPRB Buffer (ß-ME added) if plant sample
contains sticky secondary metabolites such as maize with milky
endosperm or mycelia of filamentous fungi.
Note: In order to release all the RNA in the sample, it is required to
disrupt the sample completely. Different samples require different
methods (ex: disruptor equipment) to achieve complete disruption.
Filtration
centrifuge
3. Incubate at 70 °C for 10 min, vortex every 3 min during incubation.
Total RNA Binding
centrifuge
4. Place a Filter Column to a 50 ml centrifuge tube (not provided).
And transfer the entire sample mixture to the Filter Column.
5. Centrifuge at full speed (4500~6,000 rpm) for 5 min at 4 °C.
centrifuge
Washing (Wash1)
(Wash2)
Total RNA Elution
centrifuge
6. Transfer the clarified flow-through to a new 50 ml centrifuge tube (not
provided) and adjust the volume of the clarified flow-through.
--Avoid pipett any debris and pellet when transfering the clarified
flow-through.
(RNase-free Water)
7. Add 1 volume of 70 % ethanol to the clarified flow-through and mix
well by plus-vortexing for 5 seconds.
--For example, add 4.5 ml of 70 % ethanol to 4.5 ml of clearified
flow-through.
3
4
FavorPrep Plant Total RNA
Mini Kit (for Woody Plant)
TM
FavorPrep Plant Total RNA Mini Kit
Cat.: FAPRK 003 (50 Preps)
(for woody plant)
FAPRK 003-1 (100 Preps)
(For Research Use Only) v.1005
Kit Contents
FAPRK 003
(50 preps)
FARB-1 Buffer
30 ml
60 ml
FARB-2 Buffer
4 ml
8 ml
Wash Buffer 1
30 ml
60 ml
Wash Buffer 2 (conc.)*
15 ml
35 ml
RNase-free water
6 ml
6 ml
FARB Mini Column
50 Pcs
100 Pcs
2ml Collection Tube
50 Pcs
100 Pcs
FAPRK 003-1
(100 preps)
Brief Procedure
Grind plant sample in liquid nitrogen
Lysis
(FARB-1 Buffeer)
(FARB-2 Buffeer , 70 °C)
centrifuge
RNA Binding
centrifuge
Wash (Wash1)(Wash2)
centrifuge
RNA Elution
*Add 60 ml / 140 ml ethanol (96~100%) to Wash Buffer
centrifuge
when first open.
Specifications
7
Sample Amount: up to 100 mg plant tissue or 1 X 10 plant cells
Operation time: About 30~60 min
Binding Capacity: up to 100 µg total RNA
Expected Yield: up to 5~30 µg total RNA from young leave
Elution volume: 50 µl
Important Notes
1. Make sure everything is RNase-free when handling RNA.
2. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers.
3. Pipet a required volume of FARB-1 Buffer to another RNase-free container and add 10 µl of
ß-mercaptoethanol (ß-ME) per 1ml FARB-1 Buffer before use.
4. Add required volume of ethanol(96-100%) as bottle indicated to Wash Buffer 2 when first open.
5. Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10mM MnCl2, 20mM Tris-HCl, pH7.0 at 25 °C)
to final conc.= 0.5U/µl.
General Protocol:
Please Read Important Notes Before Starting Following Steps.
1.Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and transfer to a new
microcentrifuge tube (not provided).
2.Add 500 µl of FARB-1 Buffer (ß-ME added) to the sample powder and vortex vigorously.
Note: In order to release all the RNA in the sample, it is required to disrupt the sample completely.
Different samples require different methods (ex: disruptor equipment) to achieve complete disruption.
3.Add 50 µl of FARB-2 Buffer and incubate at 70 °C for 10 min, vortex every 3 min during incubation.
1
4. Centrifuge at 12,000 rpm for 5 min at 5 °C.
5.Transfer the clarified supernatant to a new microcentrifuge tube (not provided) and adjust the volume of
the clear lysate.
--Avoid pipett any debris and pellet from the Collection Tube.
6.Add 0.9 volume of ethanol (96-100%) to the clear lysate and mix well.
--For example, add 450 µl of ethanol to 500 µl of clear lysate.
7.Place a FARB Mini Column into a Collection Tube, and transfer 750 µl of the ethanol added sample mixture
(including any precipitate) to FARB Mini Column. Centrifuge at full speed (14,000 rpm or 10,000 x g) for 1 min
and discard the flow-through.
8.Repeat step 7 for rest of the sample mixture.
9.(Optional):To eliminate genomic DNA contamination of RNA, follow the steps from 9a. Otherwise, proceed
to step 10 directly.
9a. Add 250 µl of Wash Buffer 1 to FARB Mini Column, Centrifuge at full speed (14,000 rpm or 10,000 x g) for
1 min then discard the flow-through.
9b. Add 60 µl of RNase-free DNase 1 solution (0.5 U/µl, not provided) to the membrane center of FARB Mini
Column. Place the Column on the benchtop for 15 min.
9c. Add 250 µl of Wash Buffer 1 to FARB Mini Column. Centrifuge at full speed (14,000 rpm or 10,000 x g) for
1 min then discaard the flow-through.
9d. After DNase 1 treatment, proceed to step 11.
10. Add 500 µl of Wash Buffer 1 to wash FARB Mini Column, Centrifuge for 1 min then discard the flow-through.
11.Wash FARB Mini Column twice with 750 µl of Wash Buffer 2 by centrifuge at full speed (14,000 rpm or 10,000
x g) for1 min then discard the flow-through.
--Make sure that ethanol has been added into Wash Buffer 2 when first open.
12.Centrifuge at full speed (14,000 rpm or 10,000 x g) for an additional 3 min to dry the column.
--Important Step! This step will avoid the residual liquid to inhibit subsequent enzymatic reaction.
13.Place FARB Mini Column to Elution Tube.
14. Add 50 µl of RNase-free ddH2O to the membrane center of FARB Mini Column. Stand FARB Mini Column
for 1 min.
--Important Step! For effective elution, make sure that the elution solution is dispensed of the membrane
center and is absorbed completely.
15. Centrifuge at full speed (14,000 rpm or 10,000 x g) for 2 min to elute RNA.
16. Store RNA at -70 °C.
2
TM
FavorPrep Plant Total RNA Mini Kit (Sample)
Cat.: FAPRK 000-Woody, 4 Preps
(for woody plant)
(For Research Use Only) v.1305
Kit Contents
Brief Procedure
FAPRK 000-Woody
(4 Preps)
Grind plant sample in liquid nitrogen
FARB-1 Buffer
1.5 ml x2
FARB-2 Buffer
0.5 ml
Wash Buffer 1
1.5 ml x2
Wash Buffer 2 (conc.)*
1.5 ml
RNase-free water
0.5 ml
FARB Mini Column
4 pcs
2 ml Collection Tube
4 pcs
Lysis
(FARB-1 Buffeer)
(FARB-2 Buffeer , 70 °C)
centrifuge
RNA Binding
centrifuge
Wash (Wash1)(Wash2)
centrifuge
RNA Elution
*Add 6 ml ethanol (96~100%) to Wash Buffer when first open.
centrifuge
Specifications
7
Sample Amount: up to 100 mg plant tissue or 1 X 10 plant cells
Operation time: About 30~60 min
Binding Capacity: up to 100 µg total RNA
Expected Yield: up to 5~30 µg total RNA from young leave
Elution volume: 50 µl
Important Notes
1. Make sure everything is RNase-free when handling RNA.
2. Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers.
3. Pipet a required volume of FARB-1 Buffer to another RNase-free container and add 10 µl of
ß-mercaptoethanol (ß-ME) per 1ml FARB-1 Buffer before use.
4. Add required volume of ethanol(96-100%) as bottle indicated to Wash Buffer 2 when first open.
5. Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10mM MnCl2, 20mM Tris-HCl, pH7.0 at 25 °C)
to final conc.= 0.5U/µl.
General Protocol:
Please Read Important Notes Before Starting Following Steps.
1.Grind up to 100 mg plant sample under liquid nitrogen to a fine powder and transfer to a new
microcentrifuge tube (not provided).
2.Add 500 µl of FARB-1 Buffer (ß-ME added) to the sample powder and vortex vigorously.
Note: In order to release all the RNA in the sample, it is required to disrupt the sample completely.
Different samples require different methods (ex: disruptor equipment) to achieve complete disruption.
3.Add 50 µl of FARB-2 Buffer and incubate at 70 °C for 10 min, vortex every 3 min during incubation.
1
4. Centrifuge at 12,000 rpm for 5 min at 5 °C.
5.Transfer the clarified supernatant to a new microcentrifuge tube (not provided) and adjust the volume of
the clear lysate.
--Avoid pipett any debris and pellet from the Collection Tube.
6.Add 0.9 volume of ethanol (96-100%) to the clear lysate and mix well.
--For example, add 450 µl of ethanol to 500 µl of clear lysate.
7.Transfer 750 µl of the ethanol added sample mixture (including any precipitate) to FARB Mini Column Set.
Centrifuge at full speed (14,000 rpm or 10,000 x g) for 1 min and discard the flow-through.
8.Repeat step 7 for rest of the sample mixture.
9.(Optional):To eliminate genomic DNA contamination of RNA, follow the steps from 9a. Otherwise, proceed
to step 10 directly.
9a. Add 250 µl of Wash Buffer 1 to FARB Mini Column, Centrifuge at full speed (14,000 rpm or 10,000 x g) for
1 min then discard the flow-through.
9b. Add 60 µl of RNase-free DNase 1 solution (0.5 U/µl, not provided) to the membrane center of FARB Mini
Column. Place the Column on the benchtop for 15 min.
9c. Add 250 µl of Wash Buffer 1 to FARB Mini Column. Centrifuge at full speed (14,000 rpm or 10,000 x g) for
1 min then discaard the flow-through.
9d. After DNase 1 treatment, proceed to step 11.
10. Add 500 µl of Wash Buffer 1 to wash FARB Mini Column, Centrifuge for 1 min then discard the flow-through.
11.Wash FARB Mini Column twice with 750 µl of Wash Buffer 2 by centrifuge at full speed (14,000 rpm or 10,000
x g) for1 min then discard the flow-through.
--Make sure that ethanol has been added into Wash Buffer 2 when first open.
12.Centrifuge at full speed (14,000 rpm or 10,000 x g) for an additional 3 min to dry the column.
--Important Step! This step will avoid the residual liquid to inhibit subsequent enzymatic reaction.
13.Place FARB Mini Column to Elution Tube.
14. Add 50 µl of RNase-free ddH2O to the membrane center of FARB Mini Column. Stand FARB Mini Column
for 1 min.
--Important Step! For effective elution, make sure that the elution solution is dispensed of the membrane
center and is absorbed completely.
15. Centrifuge at full speed (14,000 rpm or 10,000 x g) for 2 min to elute RNA.
16. Store RNA at -70 °C.
2