Allele Biotech-Introducing Cost Effectiveness to Research
T
he Allele RNA Purication Kit is designed for the purication of high-quality RNA from total RNA or RNA enzymatic reactions, such as in vitro transcrip-tion. This system employs modied silica membranes as selective nucleic acid captures in a spin-column format. RNA molecules (ssRNA > 80 bases, dsRNA > 150 bps), including RNAs from in vitro transcription re-action, capped RNAs, amino allyl-modied RNAs, Bi-otin and Cy-dye labeled RNAs can specically interact with the modied silica membrane in the RNA Bind-ing Buffer, while nucleotides, short oligonucleotides, salts, proteins and other contaminants are not bound and remain in the solution which is passed through. After washing the column with washing buffer to re-move unbound material, the puried RNA can be eas-ily eluted in 10 mM Tris Elution Buffer or water. Puri-ed RNA can be used for any downstream application that requires high quality RNA, such as qRT-PCR, mi-croarray, RNA-seq, RNase protection assay and other transcriptional RNA analysis.
T
he components included with
RNA Purication System
are listed above. Upon receipt, store all components at room temperature with the excep-tion of RNA Binding Buffer (RB4), which needs to be stored at 4 ° C..
♦
Fast and Easy Protocol:
The procedure consists of three steps: RNA binding to membrane, contaminant being washed away and RNA elution from membrane. It takes less than 15 minutes to nish the purication procedure.
♦High Yield and Quality:
The modified membrane and RNA binding buffer system provides for maximum RNA capturing and release. The recovery is more than 75% of input RNA of high purity with efficient removal ofnucleotides, short oligonucleotides, salts and pro-teins.
♦
Technical data:
RNA length of ssRNA > 80 bases or dsRNA > 150 bps is recommended for the procedure. RNA input from 1 μg to 300 μg can be puried with each purication.
♦
This kit is for research use only. All due care and at-tention should be exercised in the handling of the kits.
♦
Wear a laboratory coat, disposable gloves, and eye protection when handling reagents and tubes. Avoid ingestion and inhalation of reagents. In case of con-tact, wash thoroughly with water. See Material Safety Data Sheets (MSDS) for emergency procedures in case of accidental contact or ingestion. MSDS infor-mation is available upon request.
♦
Always use proper aseptic techniques to avoid nu-clease contamination when working with RNA. Use only sterile, new pipette tips to prevent cross contami-nation.
WB2:
For ABP-PP-RNAPUR025 kit, add 20 ml of 100% ethanol to Washing Buffer II (WB2) and mix well. For ABP-PP-RNAPUR050 kit, add 40 ml of 100% ethanol to Washing Buffer II (WB2) and mix well. Mark bottle that ethanol has been added.
♦
Store at room temperature and use Washing Buffer II (WB2) containing ethanol within six (6) months.
♦
Avoid nuclease contamination and clean lab bench with RNase inactivating agents such as RNase-Off or RNase-Away and wipe with 75%ethanol.
♦
Set a water baths or heat blocks at 94 °C.
RNA Purifcation System
, Employs modied silica membranes as selective nucleic acid captures in a spin-column format.
Box 1 | Contents
Kit Contents
ABP-PP-RNAPUR025ABP-PP-RNAPUR050
# of Purications
2550
CC1:
Spin-Columns with Collection Tubes
25 50
RT1:
Recovery Tubes
2550
RB4:
RNA Binding Buffer
5.0 mL10 mL
WB2:
Washing Buffer II
5.0 mL10 mL
EB3:
Elution Buffer
6.25 mL12.5 mL
Additional Materials Needed
•100% Ethanol (ACS grade or better)•Pipettor and RNase-free tips•Benchtop Centrifuge and Vortexer •Nuclease-free 1. 7 ml microcentrifuge tubes•Heating block or water bath
FeaturesGeneral PrecautionsPreparation
Allele Biotech-Introducing Cost Effectiveness to Research
The protocol below is for the purication of RNA from 50 μl reaction. The purication procedure may be scaled from 10-100 μl by proportionately adjusting all reagents throughout the procedure.
Binding RNA to Spin Column1.
Bring the RNA sample to 50μl with Elution Buffer (EB3) and mix well.
2.
Add 170μl RNA Binding Buffer (RB4) to the sample and mix well by pi petting solution up and down 7- 8 times to yield a homogenous solution.
3.
Add 130μl 100% ethanol to the sample and mix well by pipetting solution up and down 7-8 times to yield a homogenous solution.
4.
Transfer 350μl of the sample solution from step above to a Spin-Column with a Collection Tube (CCI).
5.
Centrifuge the Spin-Column at 8000 x g for one minute at room temperature. Discard the ltrate in the Collection Tube and reinsert the Spin-Column back into the Collection Tube.
Washing RNA Products1.
Add 400 μl of Wash Buffer II (WB2) with ethanol to the Column.
2.
Centrifuge the Spin-Column at 8000 g for 1 minute at room temperature. Discard the ltrate.
3.
Repeat step 1 and 2 above for a total of two wash-es. Discard the ltrate.
4.
Centrifuge the spin-column at maximum speed (10000- 15000 g, or 10000- 14000 rpm) for extra 1 minute to remove any residual wash buffer with etha-nol. Discard Collection Tube.
5.
Reinsert the Spin-Column into a new clean micro-centrifuge Recovery Tube (RTI).
Eluting RNA Products1.
Preheat 200-1000μl Elution Buffer (EB3) in a 1.7 ml tube to 94 °C with a heat block or a water bath.
2.
Add 50μl pre-heated Elution Buffer (EB3) into the center of the Spin-Column.
3.
Incubate the column for 1 minute at room tempera-ture.
4.
Centrifuge the column at maximum speed for 1 min-ute at room temperature. The Recovery Tube (RTI) contains puried RNA.
5.
To maximize RNA recovery, you may perform a sec-ond elution with 50μl pre-heated Elution Buffer (EB3) with the same recovery tube, if desired. This can in-crease the total RNA yield by 10-30 %, but the nal concentration of puried RNA in the recovery tube is reduced because of the increased volume.
6.
Store the puried RNA on ice. At this point, samples are ready for desired downstream application such as qRT-PCR, or store the RNA samples at -80 °C for long-term storage until further use.
Electrophoresis and Downstream Application
Puried RNA can be examined by agarose gel electro-phoresis. Yield can be measured with a spectropho-tometer at 260 nm, uorescent RNA assays or other quantication methods by diluting an aliquot of the pu-ried RNA sample (usually 1:50- 1:200 dilution). For electrophoresis, loading 3-8 μl puried RNA is recom-mended. The puried RNA is suitable for use in qRT-PCR, microarray, RNA-seq, RNase protection assay and other transcriptional RNA analysis.
Protocols
Allele Biotech-Introducing Cost Effectiveness to Research
Box 2 | Troubleshooting
Problem
Cause
Solution
Low product yield
Amount of RNA used was less than recommended or poor quality of starting materialCheck original RNA with gel electropho-resis or Agilent 2100
OR
Add additional RNA sample. Incomplete MixingMix RNA sample with RNA Binding Buffer and 100% Ethanol thoroughly before loading onto spin-columnNuclease contaminationMake sure lab bench and pipettes are clean and RNase-free. Wear gloves at all times during the whole procedure and change gloves frequently to protect reagents and RNA from nuclease that are present on skin. Use RNase-free pipette tips and tubes to handle all solu-tions; avoid used tips or used tubes for reagents
No RT-PCR product
Missing Component in the RT-PCR mixture Be sure to add all components. Check positive and negative controls for RT-PCR reaction.
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