Molecular Diagnostic Products – Instruction Manuals
1. Fast RNA Stain. Instruction Manual
2. Fast RNA and DNA Elution. Instruction Manual
1. Fast RNA Stain. Instruction Manual.
Detection of RNA and single stranded DNA in polyacrylamide gels. RNA and DNA bands stained using this protocol can be detected within 5-10 minutes after adding Fast RNA Stain™.
- Turn the power supply off after electrophoresis, separate the glass plates carefully and cover only the polyarylamide gel with Fast RNA StainT solution. Incubate the gel for 2-5 minutes at room temperature.
- Pour out the stain solution and cover the gel with autoclaved water. Incubate for 5 minutes. Pour out the water. Add fresh autoclaved water. Continue destaining until blue bands and clear background are obtained. RNA and DNA bands will become visible within 5-10 minutes, depending on gel thickness.
- Detected nucleic acids can be eluted from gel by Fast RNA and DNA Elution Kit™ from polyacrylamide gels, and used for RT-PCR, sequencing and labeling reactions.
- Alternatively, RNA and DNA can be fixed on gel for a future record. Place gel on two sheets of Whatman 3MM filter paper and cover top with plastic wrap. Dry the polyacrylamide gel in a conventional gel dryer for 1 hour at 80°C.
Detection of RNA in agarose gels.
- Place an agarose gel in a glass or plastic container. Rinse the formaldehyde from the gel to prevent high background staining by soaking it twice for 20 minutes in 0.5M ammonium acetate. Add Fast RNA StainT and agitate slowly for 5-10 minutes, depending on gel thickness.
- Pour out the stain solution and wash gel twice with autoclaved water for 10 minutes with slow agitation. Pour out the water. Add fresh autoclaved water and shake gently until a clear background is obtained.
- After confirmation of the presence of RNA on an agarose gel, use the gel directly for Northern blotting or store in plastic wrap at 4°C.
Detection of RNA and DNA on hybridization membranes.
- Immobilize nucleic acids on the hybridization membrane by UV cross-linking on a transilluminator or bake it for 2 hours at 80°C (see the manufacturer’s directions).
- Stain the membrane with Fast RNA Stain™ solution for 1 minute at room temperature. Pour out the stain solution and rinse the membrane briefly with autoclave water. RNA and DNA bands stain blue against the white to the bluish background of the membrane. Membrane can be used immediately for hybridization or stored in plastic wrap at 4°C.
STORAGE CONDITIONS FOR THE PRODUCT:
Store Fast RNA Stain\u2122 Kit at room temperature. The product is stable for at least one year after the date of purchase.
2. Fast RNA and DNA Elution Kit™. Instruction Manual.
Nucleic acids are commonly purified further by one of three procedures: centrifugation, column chromatography or gel electrophoresis. The method of choice is usually gel electrophoresis because of its high resolution capacity. At the same time, the elution of RNA from a polyacrylamide gel is a more difficult part. HealthGene is offering the fastest and easiest protocol for RNA and DNA elution from the gel using Fast RNA and DNA ElutionT Kit™.
Elution of RNA and DNA from a polyacrylamide gel:
- After a polyacrylamide gel electrophoresis, disconnect the power supply, separate the glass plates and locate RNA or DNA bands using Fast RNA Stain Kit .
- Cut the band from the gel and elute RNA or DNA by crushing the gel into small pieces in 1.5 ml microcentrifuge tube with 400 µl of Solution A from Fast RNA and DNA Elution Kit™. Incubate the tube at room temperature for 30 minutes with occasional vortexing. Remove the gel debris by centrifugation for 10 minutes at 12,000 rpm. Transfer the supernatant to a new 1.5 ml tube.
- Precipitate RNA or DNA for 2 hours or longer at \u201320°C using 1 ml of Solution B from Fast RNA and DNA Elution Kit™ and collect RNA or DNA by centrifugation at 12,000 rpm for 15 minutes.
- Determine the amount of RNA or DNA present by dissolving it in 50-100 µl of autoclaved water and determining the absorbency at 260 nm (1 A260 unit = approximately 40 µg of RNA or 50 µg of DNA).
- For future use of RNA and DNA samples, store them at -20°C.
STORAGE CONDITIONS FOR THE PRODUCT:
Store Fast RNA and DNA Elution Kit ™ at room temperature. The product is stable for at least one year after the date of purchase.