The kit is also suitable for isolation of total RNA from cultured cells, tissues, and bacteria. This procedure has been tested for isolating nucleic acids from Hepatitis A, Hepatitis C and HIV. The isolated DNA/RNA can be used for PCR, RTPCR and other downstream applications
Purified DNA is suitable for PCR, restriction digestion, and hybridization techniques
- Add 200 mL 100 % ethanol to RNA Wash Buffer before use.
- Add all Carrier Nucleic acid to Buffer HLY. Transfer Carrier Nucleic acid to the bottle of HLY Lysis Buffer. Store at 4 °C. Warm up Buffer HLY/carrier before use if precipitation forms at 4 °C.
- Prepare Wash Buffer and HLY Lysis Buffer/carrier Nucleic acid solution as instructed
- Add 150 μL plasma, acellular body fluid, cell culture supernatant to each well of a 1.2 mL 96-well plate (not provided).
- Add 10 μL Protease K to each well.
- Add 500 μL HLY Lysis Buffer/Carrier Nucleic acid solution to each well. Seal the plate with a Sealing Film.
- Keeping the 96-well plate flat on the bench, shake vigorously back and forth for 30 seconds. Rotate the plate 90° and shake the plate for another 30 seconds.
- Let sit at room temperature for 10 minutes.
- Briefly centrifuge at 500g to collect any liquid droplets from the film.
- Remove and discard the Sealing Film.
- Add 350 μL 100 % ethanol to each well. Seal the plate with a Sealing Film.
- Vortex the plate for 30 seconds. Briefly centrifuge at 500g to collect any liquid droplets from the film. Remove and discard the Sealing Film.
- Place a 96 Well Plate onto a 96-well Deep Plate (provided).
- Transfer 500 μL sample from Step 8 (including any precipitate that may have formed) to each well of the 96 Well Plate.
- Seal the 96 Well Plate with a Sealing Film.
- Centrifuge at 4,000g for 5 minutes at room temperature.
- Discard the filtrate from the 96-Well Deep Plate.
- Remove and reuse the Sealing Film in the following step.
- Repeat Steps 11-15 until the entire sample has been transferred to the 96 Well Plate.
- Remove the Sealing Film.
- Add 750 μL Wash Buffer to each well. Seal the plate with the Sealing Film. Place the 96 Well Plate onto the 96-well Deep Well Plate.
- Centrifuge at 4,000g for 10 minutes at 4 °C. Discard the flow through and centrifuge the plate at 4000xg for 10 min at 4 °C.Note: It is important to dry the 96 Plate matrix before elution. Residual ethanol may interfere with downstream applications.
- Remove and discard the Sealing Film. Transfer the 96 Well Plate to an Elution Plate.
- Add 100-150 μL DEPC-treated water to each well. Seal the plate the new Sealing Film. Make sure to add water directly onto RNA matrix.
- Let sit for 1 minute at room temperature.
- Centrifuge at 4,000g for 5 minutes at 4 °C. The RNA/DNA is in the flow through.
- Store RNA/DNA at -80 °C.
For Research Use only