INSTRUCTION MANUAL RNA Clean & Concentrator -100

INSTRUCTION MANUAL
RNA Clean & Concentrator™-100
Catalog No. R1019
Highlights

Quick (15 minute) method for cleaning and concentrating RNA.

Ideal for the purification of RNA from aqueous phase following acid phenol extraction.

Fast-Spin column technology allows RNA to be eluted into minimal volumes (≥100 µl).

Eluted RNA is pure and ready for subsequent analysis and molecular manipulation.
Contents
Product Contents .................................................. 1
Specifications ....................................................... 1
Product Description .............................................. 2
Buffer Preparation ................................................ 3
Protocol ................................................................ 3
Ordering Information ............................................ 4
Appendices ........................................................... 5
Related Products .................................................. 6
For Research Use Only
Ver. 1.0.1
ZYMO RESEARCH CORP.
Toll Free: 1-888-882-9682  Fax: 1-714-288-9643  Web: www.zymoresearch.com  E-mail: [email protected]
Page 1
Satisfaction of all Zymo
Research products is
guaranteed. If you should
be dissatisfied with this
product please call 1-888882-9682.
Product Contents
RNA Clean & Concentrator™-100
Kit Size
R1019
(25 Preps.)
Storage
Temperature
RNA Binding Buffer
100 ml
Room Temp.
RNA Prep Buffer
10 ml
Room Temp.
RNA Wash Buffer1 (concentrate)
6 ml
Room Temp.
DNase/RNase-Free Water
10 ml
Room Temp.
Zymo-Spin™ V-E Columns w/ Reservoir
25
Room Temp.
Collection Tubes
50
Room Temp.
Instruction Manual
1
-
Note - Integrity of kit components is guaranteed for up to one year from date of purchase. Reagents are routinely tested
on a lot-to-lot basis to ensure they provide the highest performance and reliability.
1
Add 24 ml 100% ethanol (26 ml 95% ethanol) to the 6 ml RNA Wash Buffer concentrate before use.
Specifications

Sample Sources – modified, labeled, impure RNA, DNase treated RNA, in vitro
transcription products, RNA from aqueous phase following acid phenol extraction
methods (e.g., TRI Reagent®; see Appendix A).

Size Limit – RNA ≥17 nucleotides (or ≥200 nt; see Appendix C).

Format – Spin column

RNA Purity – High quality RNA (A260/A280 >1.8, A260/A230 >1.8) suitable for all
downstream RNA-based manipulations.

RNA Recovery – Up to 125 µg RNA can be eluted into ≥100 µl RNase-free water
allowing for a highly concentrated sample.

RNA Storage – RNA is eluted with RNase-free water and can be stored at ≤-70 ºC.
The addition of RNase inhibitors to the RNA is recommended for prolonged storage.

Equipment Needed – Vacuum manifold, microcentrifuge.
Note - ™ Trademarks of Zymo Research Corporation. This product is for research use only and should only be used by trained
professionals. It is not intended for use in diagnostic procedures. Some reagents included with this kit are irritants. Wear protective gloves
and eye protection. Follow the safety guidelines and rules enacted by your research institution or facility. TRI Reagent® is a registered
trademark of Molecular Research Center.
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 2
Product Description
The RNA Clean & Concentrator™-100 provides a simple and reliable method for the
rapid preparation of up to ~125 µg of high-quality RT-PCR-ready RNA. This simple
procedure is based on the use of a unique single-buffer system and Fast-Spin column
technology. The procedure is easy: just add the buffer to your sample, adjust the
conditions for binding by adding ethanol, and the RNA is then concentrated into ≥100 µl
of RNase-free water using a Zymo-Spin™ V-E Column. RNA fragments (≥17 bases)
can be safely treated and recovered using this kit. The result is highly-concentrated,
purified RNA that is suitable for subsequent RNA-based methods including RT-PCR,
hybridization, etc.
For Assistance, please
contact Zymo Research
Technical Support at
1-888-882-9682 or e-mail
[email protected].
The entire procedure typically takes about 15 minutes.
Concentration of diluted RNA samples (n = 3, total input
= 1 μg RNA) using the RNA Clean & Concentrator™-5.
RNA was eluted with 20 μl RNase-free water.
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 3
Make sure guidelines are
followed to ensure the RNA
isolation procedure is
performed in an RNase-free
environment.
Buffer Preparation
Before starting, add 24 ml 100% ethanol (26 ml 95% ethanol) to the 6 ml RNA Wash
Buffer concentrate.
Protocol
Notes:
All centrifugation steps should be performed at 10,000 – 16,000 x g.
RNA species ≥17 nt will be recovered1.
1
Alternatively, to eliminate
tRNA and other small RNA
species up to 200 nt, see
Appendix C (page 5).
2
Set vacuum source at
≥500 mm Hg.
1.
Add 2 volumes of RNA Binding Buffer to each volume of RNA sample (e.g., 1 ml
buffer to 0.5 ml RNA sample) and mix well.
2.
Add one volume 95-100% ethanol to the mixture (e.g., 1.5 ml ethanol to 1.5 ml
sample-buffer mixture) and mix well.
3.
Transfer the sample mixture into the Zymo-Spin™ V-E Column w/ Reservoir
mounted on a vacuum manifold and start vacuum2.
4.
Remove the reservoir and transfer the Zymo-Spin™ V-E Column into a
Collection Tube. Centrifuge the column for 30 seconds.
At this point, samples can be in-column DNase treated (Appendix B, page 5).
3
To maximize RNA yield,
increase the elution volume
and/or repeat the elution.
5.
Add 400 µl RNA Prep Buffer to the column and centrifuge for 1 minute. Discard
the flow-through.
6.
Add 400 µl RNA Wash Buffer to the column and centrifuge for 30 seconds.
Discard the flow-through and repeat Step 6.
7.
Centrifuge the Zymo-Spin™ V-E Column in an emptied collection tube for 2
minutes to ensure complete removal of the wash buffer. Transfer the column
carefully into an RNase-free tube.
8.
Add ≥100 µl of DNase/RNase-Free Water3 directly to the column matrix and let
stand for 1 minute at room temperature. Centrifuge at top speed for 1 minute.
Eluted RNA can be used immediately or stored at ≤-70°C.
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 4
Ordering Information
Product Description
Catalog No.
Kit Size
R1015
R1016
R1017
R1018
50 Preps.
200 Preps.
50 Preps.
100 Preps.
RNA Clean & Concentrator™-100
R1019
25 Preps.
For Individual Sale
Catalog No.
Amount
R1013-2-25
R1013-2-50
R1013-2-100
R1013-2-1000
R1060-2-10
R1060-2-25
R1003-3-6
R1003-3-12
R1003-3-24
R1003-3-48
C1001-50
C1001-500
C1001-1000
W1001-1
W1001-4
W1001-6
W1001-10
25 ml
50 ml
100 ml
1000 ml
10 ml
25 ml
6 ml
12 ml
24 ml
48 ml
50
500
1000
1 ml
4 ml
6 ml
10 ml
RNA Clean & Concentrator™-5
RNA Clean & Concentrator™-25
RNA Binding Buffer
RNA Prep Buffer
RNA Wash Buffer (concentrate)
Collection Tubes
DNase/RNase-Free Water
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 5
Appendix A: RNA Recovery from Aqueous Phase after Trizol® Extraction
1. Following Trizol®/chloroform or similar2 extraction, carefully transfer the upper
aqueous phase into an RNase-free tube (not provided).
2. For each volume of the aqueous phase (as measured or estimated), add 1 volume
ethanol (95-100%) and mix.
3. Continue with purification (page 3/step 3).
Appendix B: In-column DNase digestion1
The DNase digestion procedure can be performed using the DNase I Set (E1009, not
provided) or any DNase I together with its dedicated reaction buffer. DNase I maintains
activity in the RNA Wash Buffer.
1. Following the RNA binding step (page 3/step 3), prewash the column with 400 µl
RNA Wash Buffer. Centrifuge for 30 seconds. Discard the flow through.
2. For each sample to be treated, prepare DNase I reaction mix in an RNase-free
tube (not provided). Add the reagents in the following order:
DNase I
10X Reaction Buffer
RNA Wash Buffer1 (with ethanol added)
20 µl (1 U/µl)
20 µl
160 µl
3. Add 200 µl DNase I reaction mix directly to the column matrix. Incubate at room
temperature (20-30ºC) for 15 minutes. Then centrifuge for 30 seconds.
4. Continue with wash steps (page 3/step 5).
Appendix C: Elimination of tRNA and other small RNA species up to 200 nt
1. Adjust the RNA Binding Buffer by mixing equal volumes of the buffer and ethanol
(95-100%) (e.g., 100 µl buffer and 100 µl ethanol).
2. Add 2 volumes of the adjusted buffer to each RNA sample to be treated (e.g., 200 µl
buffer to 100 µl RNA) and mix well.
3. Continue with purification (page 3/step 3).
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 6
Related Products
Product
Description
Prep/Format
Catalog
50/column
100/column
2x96/plate
50/column
200/column
2x96/plate
4x96/plate
50/column
R1020
R1021
R1022
R1034
R1035
R1040
R1041
R1039
50/column
50/column
200/column
25/column
2x96/plate
4x96/plate
R1050
R1054
R1055
R1056
R1052
R1053
50/column
200/column
R1060
R1061
50/column
200/column
50/column
R1064
R1065
R1003
R1007
Total RNA Purification
ZR Whole-Blood RNA MiniPrep
whole blood, partitioned blood
™
ZR-96 Whole-Blood RNA Kit™
plasma, serum, liquids, cells, tissue
ZR Viral RNA Kit™
™
ZR-96 Viral RNA Kit
ZR Urine RNA Isolation Kit™
urine, liquid samples
Quick-RNA™ MicroPrep
cells, tissue, buccal cells, buffy coat, plasma, serum, biological liquids
Quick-RNA™ MiniPrep
Quick-RNA™ MidiPrep
ZR-96 Quick-RNA™
cells, tissue, buccal cells, buffy coat, plasma, serum, biological liquids;
DNA removal column, small-RNA recovery (≥17nt), in-column DNase
treatment protocol
ZR RNA MicroPrep™
ZR RNA MiniPrep™
Pinpoint™ Slide RNA Isolation System Kit I
fresh tissue sections
Pinpoint™ Slide RNA Isolation System Kit II
paraffin-embedded tissue
50/column
ZR Fungal/Bacterial RNA MicroPrep™
bacteria, yeast, fungi; BashingBead™ lysis
50/column
R2010
50/column
R2014
plant parts and tissues; BashingBead™ lysis, RT/PCR inhibitor removal
50/column
R2024
ZR Tissue & Insect RNA MicroPrep
insect, small tissue samples; BashingBead™ lysis
50/column
R2030
YeaStar RNA Kit™
yeast, fungi
50/column
R1002
50/column
200/column
R1015
R1016
50/column
100/column
25/column
R1017
R1018
R1019
R1080
R1013
R1014
R1011
ZR Fungal/Bacterial RNA MiniPrep™
ZR Plant RNA MiniPrep™
™
RNA Clean-up, Concentration & Recovery
RNA Clean & Concentrator™-5
modified/labeled/impure/diluted RNA; small-RNA recovery (≥17nt); acid
phenol extracted RNA directly from aqueous phase, in-column DNase
treatment protocol
RNA Clean & Concentrator™-25
RNA Clean & Concentrator™-100
DNA-Free RNA Kit™
DNase I treatment; small-RNA recovery (≥17nt)
Zymoclean™ Gel RNA Recovery Kit
agarose gel separated RNA
2x96/plate
50/column
200/column
50/column
polyacrylamide gel separated RNA; small-RNA recovery (≥17nt)
20/column
R1070
50/column
D7001
25/column
100/columns
D7020
D7021
ZR-96 RNA Clean & Concentrator™
™
ZR small-RNA PAGE Recovery Kit
DNA/RNA Parallel Purification
™
ZR-Duet DNA/RNA MiniPrep
cells, tissue, liquids; DNA/RNA separation, small-RNA recovery (≥17nt),
in-column DNase treatment protocol
DNA/RNA Co-Purification
ZR Viral DNA/RNA Kit
plasma, serum, liquids, cells, tissue
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com