Total RNA Extraction Kit (Tissue)
No:DMRTS100 & 300
For research use only
Sample: up to 30 mg of tissue, up to 25 mg of paraffin-embedded tissue
Yield : up to 30μg
Introduction
The BioDiamond Total RNA Extraction Kit provides a fast, simple, and cost-effective method for isolation of total RNA from tissue sample. Detergents and chaotropic salt are used to lyse cells and inactivate RNase. The specialized high-salt buffering system allows RNA species bases to bind to the the glass fiber matrix of the spin column while contaminants pass through the column. Impurities are efficiently washed away, and the pure RNA is eluted with REL Buffer without phenol extraction or alcohol precipitation. RNA purified with The BioDiamond Total RNA Extraction Kit is suitable for a variety of routine applications including RT-PCR, cDNA Synthesis, Northern Blotting, Differential display, Primer Extension and mRNA Selection. The entire procedure can be completed within 25-40 minutes.
Kit Contents
NOTE
★Always wear latex or vinyl gloves while handling reagents and RNA samples to prevent RNase contamination.
★Add ethanol (96–100%) to Buffer W2, shake before use (see bottle label for volume).
★Check Buffers before use for salt precipitation. Redissolve any precipitate by warming to 37°C.
★Buffers RA and W1 contain irritants. Wear gloves when handling these buffers.
Quality Control
In accordance with FairBiotech’s ISO-certified Total Quality Management System, the quality of the BioDiamond Total RNA Isolation Kit is tested on a lot-to-lot basis to ensure
consistent product quality.
Additional requirements
*Ethanol (96~100%)
*RNase-free pipet tips and 1.5 ml microcentrifuge tubes
*14.3 M ß-mercaptoethanol
# For Optional Step (DNA Residue Degradation):
Add 2 µl DNAse I (2KU/µl) and 10 µl reaction buffer {300 mM Tris-HCl (pH 7.5), 60 mM MnCl2, 300 µg/ml BSA } to the 50μl final product. Let stand for 10 minutes at room temperature (at 37ºC).
Protocol
Step 1 Sample Preparation
Fresh or Frozen Tissue
- Cut off up to 30 mg of fresh or frozen animal tissue and grind the sample using one of the micropestles provided in a microcentrifuge tube OR under liquid nitrogen to a fine powder using a mortar and pestle. (If using frozen animal tissue, the sample MUST have been flash frozen in liquid nitrogen and immediately stored at -70ºC until use, to avoid RNA Degradation).
Additional requirements: xylene, absolute ethanol
- Slice small sections (up to 25 mg) from blocks of paraffin-embedded tissue and transfer to a 1.5 ml microcentrifuge tube.
- Add 1 ml of xylene to the tube. Vortex vigorously and incubate at room temperature for approximately 10 minutes. Vortex occasionally during incubation.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Add 1 ml of absolute ethanol to wash the sample pellet and mix by inverting.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Add 1 ml of absolute ethanol to wash the sample pellet again and mix by inverting.
- Centrifuge at 14-16,000 x g for 3 minutes. Remove the supernatant.
- Open the tube and Incubate at 37ºC for 15 minutes to evaporate any ethanol residue.
- Proceed with the Lysis Step.
- Add 400 µl of RR Buffer and 4 µl of ß-mercaptoethanol to the sample and grind the sample until it is completely dissolved. Transfer the dissolved sample to a RNase-free 1.5 ml microcentrifuge tube. then incubate at 80ºC for 20 minutes.
- Centrifuge at 16,000 x g for 3 minutes. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
Step 3 Binding
- Add 400 μl of 70% ethanol prepared in ddH2O (RNase-free and DNase-free) to the sample lysate from Step 2 and shake vigorously (break up any precipitate by pipetting).
- Place a RL Column in a Collection Tube. Apply 600μl of the mixture to the RL Column.
- Centrifuge at 14,000 x g for 1 minute. Discard the flow-through and place the RL Column in the same Collection tube. Transfer the remaining mixture to the same RL Column.
- Centrifuge at 14,000 x g for1 minute. Discard the flow-through and place the RL Column in the same Collection tube.
- Add 400 µl of W1 Buffer into the RL Column. Centrifuge at 14,000 x g for 30 seconds. Discard the flow-through and place the RL Column back into the same Collection tube.
- Add 600 µl of W2 Buffer (Ethanol added) into the RL Column. Centrifuge at 14,000 x g for 30 seconds. Discard the flow-through and place the RL Column back into the same Collection tube.
- Centrifuge at 14,000 x g again for 2 minutes to remove residual W2 Buffer.
- To elute RNA, place the RL Column in a clean 1.5 ml microcentrifuge tube.
- Add 50 μl of Pre-Heated REL Buffer to the center of each RL Column, let stand for 2 minutes, and centrifuge at 14,000 x g for 2 minutes.
Troubleshooting
dmrts100___300.pdf | |
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