Total RNA Extraction Kit (Blood/Cell/Bacteria)
No:DMRBA100 & 300
For research use only
Sample : up to 300 μl of whole blood, 107mammalian cells and 109 bacterial cells
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 whole blood, mammalian cells and bacterial cells. Detergents and chaotropic salt are used to lyse cells and inactivate RNase. The specialized high-salt buffering system further allows all RNA 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 pure RNA is eluted with REL Buffer without phenol extraction or alcohol precipitation needs. 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, shaking 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 Extraction Kit is tested on a lot-to-lot basis to
ensure consistent product quality.
Additional requirements
- Ethanol (96~100%)*1.5 ml microcentrifuge tubes
- 14.3 M ß-mercaptoethanol*RNase-free pipet tips
- 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 25ºC).
- For Gram-positive bacteria sample: lysozyme buffer (20 mg/ml lysozyme; 20 mM Tris-HCl; 2 mM EDTA; 1% TritonX-100; pH 8.0, prepare the lysozyme buffer immediately prior to use)
- For Fungus sample: lyticase or zymolase, sorbitol buffer (1.2 M sorbitol;10 mM CaCl2; 0.1 M Tris-HCl pH 7.5; 35 mM mercaptoethanol)
Protocol
Step 1 Sample Cells Harvesting
Fresh Blood
- Collect blood in EDTA-Na2 treated collection tubes (or other anticoagulant mixtures).
- Transfer up to 300 μl of blood to a sterile1.5 ml microcentrifuge tube.
- Add 900 μl of RL Buffer and mix by inversion.
- Incubate the tube on ice for 10 minutes (invert twice during incubation).
- Centrifuge for 5 minutes at 4,000 x g at 4ºC. Remove the supernatant completely and resuspend the cells in 100 µl of RL Buffer by pipetting the pellet up and down.
- Transfer cultured mammalian cells (up to 107) to a sterile 1.5 ml microcentrifuge tube.
- Centrifuge at 6,000 x g for 1 minute. Remove the supernatant completely and resuspend the cells in 100 µl of RL Buffer by pipetting the pellet up and down.
- Transfer cultured bacterial cells (up to 109) to a sterile 1.5 ml microcentrifuge tube.
- Centrifuge at 12,000 x g for 1 minute. Remove the supernatant completely and resuspend the cells in 200 µl of RO Buffer by pipetting the pellet up and down. Incubate at room temperature for 5 minutes.
- Transfer cultured bacterial cells (up to 109) to a sterile 1.5 ml microcentrifuge tube.
- Centrifuge at 12,000 x g for 1 minute. Remove the supernatant completely and resuspend the cells in 200 µl of lysozyme Buffer by pipetting the pellet up and down. Incubate at room temperature for 10 minutes.
- Transfer fungus cells (up to 108) to a sterile 1.5 ml microcentrifuge tube.
- Centrifuge at 6,000 x g for 5 minute. Remove the supernatant completely and resuspend the cells in 600 µl of sorbitol Buffer by pipetting the pellet up and down.
- Add 200 U of lyticase or zymolase. Incubate at 30ºC for 30 minutes.
- Centrifuge the mixture for 10 minutes at 2,000 x g to harvest the spheroplast. Remove the supernatant completely and resuspend the cells in 200 µl of RO Buffer by pipetting the pellet. Incubate at room temperature for 5 minutes.
Fresh Blood/Mammalian Cells
- Add 400 µl of RA Buffer and 4 µl of ß-mercaptoethanol to the resuspended cells from Step 1 and shake vigorously. Incubate at room temperature for 5 minutes.
- Centrifuge at 16,000 x g for 10 minutes. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
- Add 300 µl of RA Buffer and 3 µl of ß-mercaptoethanol to the sample lysate from Step 1 and mix by vortex. Incubate at room temperature for 5 minutes.
- Centrifuge at 16,000 x g for 10 minutes. Transfer the supernatant to a new 1.5 ml microcentrifuge tube.
- Add 500 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 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
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