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Allele Biotech-Introducing Cost Effectiveness to Research
T
he Surface Bind RNA Purication Kit is designed for the optimal purication of high-quality RNA from total RNA or enzymatic reactions, such as in vitro tran-scription. Based on Solid Surface Reversible Binding (SSRB) technology, this purication system utilizes micro-tube coated with proprietary turbo binders act-ing to selectively capture and efciently bind RNA from reaction mixtures. No spin-column, lter plates, silica membranes, or magnetic beads are needed for clean-up, also no vacuum or ltration steps are required in the purication process.
I
n the presence of Binding Buffer, RNA molecules (ssRNA > 60 bases, dsRNA > 100 bps), including RNAs from in vitro transcription reactions, capped RNAs, amino allyl-modied RNAs, Biotin and Cy-dye labeled RNAs can specically interact with the turbo binders and bind to the tubes while proteins, nucleo-tides, short oligo nucleotides, salts and other contami-nants will remain in the solution. Unbound material is then removed in subsequent washing steps. The puri-ed RNA is easily eluted in 10 mM Tris Elution Buffer or water allowing for downstream applications such as RT and other gene expression analysis directly in the same tube without sample elution.
 
Easy to handle:
 All procedures have been opti-mized with single tube for ease-of-use. You can per-form RNA purication and downstream assays all in the same tube without sample transferring.
♦Maximum recovery:
Capable of maximizing RNA recovery from original RNA sample is the unique fea-ture of the kit. Unlike conventional silica membrane or bead based purification platform, there are no void spaces in the Binding Tube. This novel solid-surface capturing technology prevents washing buffer carry-over and sample trapping problems associated with membrane or bead-based column/plate purification methods. This feature not only allows fast buffer wash-ing and easy sample elution, but also provides maxi-mum sample capture and release. RNA molecules as few as 100 ng can be efficiently isolated with over 80% recovery efficiency.
 
Technical data:
RNA length of ssRNA > 60 bases or dsRNA > 100 bps is recommended for the procedure. RNA input from 100 ng to 500 μg can be puried with each purication.
T
he components included with
Surface Bind RNA Purication Kit
 are listed below. Upon receipt, store all components at room temperature.
 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.
Surface Bind RNA Purication
,  A whole new approach to RNA purifcation using Surface Bind tecnology
Box 1 | Contents
 
Kit Contents
ABP-PP-SBRNA025ABP-PP-SBRNA050# of Purications
2550
BT5:
 
Binding Tubes
25 50
RB7:
 
Binding Buffer 
 
1.0 mL2.0 mL
WBlR:
 
Washing Buffer IR
1.0 mL2.0 mL
WB2:
 
Washing Buffer II
2.5 mL5.0 mL
EB3:
 
Elution Buffer
2.2 mL4.4 mL
Additional Materials Needed
 
•100% Ethanol and Isopropanol (ACS grade or better)•Single-channel pipettor and RNase-free tips•Benchtop Centrifuge
FeaturesGeneral Precautions
 
Allele Biotech-Introducing Cost Effectiveness to Research
WBIR:
 For ABP-PP-SBRNA025 kit, add 4.0 ml of 100% ethanol to Washing Buffer IR (WBIR) and mix well. For ABP-PP-SBRNA050 kit, add 8.0 ml of 100% ethanol to Washing Buffer IR (WBIR) and mix well. Mark bottle that ethanol has been added.
Store at 4 ° C and use Washing Buffer (WBIR) con-taining ethanol within six (6) months.
WB2:
 For ABP-PP-SBRNA025 kit, add 10 ml of 100% ethanol to Washing Buffer II (WB2) and mix well. For  ABP-PP-SBRNA050 kit, add 20 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.
RB7:
 Prepare fresh working Binding Buffer (RB7) each time prior to performing RNA isolation procedure based on the number of samples processed. To make fresh working Binding Buffer (RB7), for each 10 μl of Binding Buffer (RB7) add 40 μl of 100% isopropanol. Mix well. Prepare a master working Binding Buffer so-lution based on (1) the number of samples processed and (2) any anticipated loss, generally 10 %, during dispensing. Dispense 100 μl Binding Buffer containing isopropanol per 50-μl RNA sample. Discard the un-used Binding Buffer at the end of the day.
If desired, you may add an internal control into the purication procedure. Internal control RNA should be added together with the binding buffer.
The protocol below is for the purication of RNA from 50 μl reaction. The purication procedure may be scaled from 10-100 μl by proportionately adjusting all reagents throughout the procedure.
Binding RNA Products1.
 Transfer 50 μl RNA reaction solutions to a Binding Tube (BT5). (If sample is not exactly 50 l, adjust all reagents proportionately throughout the procedure)
2.
 Add 100 μl fresh working Binding Buffer (RB7) con-taining isopropanol to the Binding Tube (BT5). Mix well with the RNA sample by pipetting up and down the solution 10 times to obtain a homogenous solu-tion.
3.
 Close the Binding Tube (BT5) cap.
4.
 Centrifuge the Binding Tube at maximum speed (about 13000 rpm or 16000 X g) at room temperature for 8 minutes to bind the RNA.
5.
 Open the cap.
6.
 Remove the solution by aspirating the solution from the exact center of the tube bottom with a pipette tip. Be sure not to scrape the walls of the tube with pipette tips during aspiration as the products are bound to the walls of the Binding
Tube.
Washing RNA Products1.
 Add 150 μl Wash Buffer IR (WB I R) containing eth-anol to each tube. Mix by pi petting solution up and down 4-5 times.
2.
 Remove the Wash Buffer IR (WBIR) from the Bind-ing Tube using the methods suggested in step 6 of “Binding RNA Products”.
3.
 Add 200 l Wash Buffer II (WB2) containing ethanol to each well. Mix by pipetting solution up and down 4-5 times.
4.
 Remove the Wash Buffer II (WB2) from the Binding Tube using the methods suggested in step 6 of “Bind-ing RNA Products”.
5.
 Repeat step 3 and 4 above for a total of two washes with Washing Buffer II (WB2).
6.
 After the nal wash, to ensure complete removal of Washing Buffer, you may spin the Binding Tube very briey after removing the majority of solution and as-pirate the last drop of liquid at the bottom center of the tube with a pipet tip; or you may invert the tube and tap the tube on a stack of clean, absorbent paper gen-tly and air-dry the Binding Tube in a lab hood for 8 - I 0 minutes to remove any residual liquid. Alternatively, put the tube in a 62°C air incubator or a hot blocker and air-dry the tube without cover for 2-4 minutes.
Eluting RNA Products
RNA attached to the walls of the Binding Tube can be stored directly in the well at -80 °C for long-term stor-age until further use. If the RNA is to be eluted, follow the procedure below.
1.
 Add 50- 80 μl Elution Buffer (EB3) into each tube.
2.
 Close the tube and vortex the tube for 10-30 sec-onds, or tap the Binding Tube (BT5) 10-15 times with a nger to allow the Elution Buffer (EB3) contacting the wall of the bottom of the Binding Tube.
3.
 Briey centrifuge the tube to collect all solution, and place the tube on ice for downstream applications. If you are not going to analyze the samples immediately, store the tube at -80 °C until use.
Electrophoresis and Downstream Application
Puried 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 quantication methods by diluting an aliquot of the pu-ried RNA sample (usually 1:50- 1:200 dilution). For electrophoresis, loading 3-8 μl puried RNA is recom-mended. The puried RNA is suitable for use in qRT-PCR, microarray, RNA-seq, RNase protection assay and other transcriptional RNA analysis.
PreparationProtocols
 
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Box 2 | Troubleshooting
 
Problem CauseSolution
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 fresh prepared RNA Binding Buffer (RB7) thoroughly before centrifuge.Low centrifugation forcesMake sure the Binding Tube was spun at 16000 x g for 8 minutes. If lower speed is used, increase the spin time.Pipette tips scrape walls of the Binding Tube too much during aspiration of solutionManeuver the pipette tip to the exact center of the bottom of the Binding Tube during aspiration. If possible, use a nar-row gel-loading pipette tip for aspiration to reduce the scrapping.Nuclease 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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