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Commit 70c9d318 authored by Gesa Helmsorig's avatar Gesa Helmsorig
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added protocols

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TRIzol RNA extraction
- Place 2 3mm metal beads into 2.0 ml tube
- Collect samples directly into the tubes on liquid nitrogen
- Store samples at -80°C until needed
- Grind your samples 2 x 45sec at maximum frequency (30 Hertz) in TissueLyser (QIAGEN), use pre-cooled adapters
- Add 500 µL of TRIzol to the frozen samples, close tubes and mix vigorously until the sample is homogeneous
- Incubate the samples for 5 min at room temperature
- Briefly spin down the samples
- Add 100 µl of chloroform, close the tubes and shake tubes 15-20 sec vigorously by hand
- Incubate the samples for 3 min at room temperature
- Centrifuge the samples at 10.000 x g for 15 min at 4°C
- Transfer 150-300 µl of the clear, aqueous, upper phase to new tubes
- Add same volume of isopropyl alcohol, gently invert 8x, do not vortex
- Incubate the samples for 10 min at room temperature
- Centrifuge the samples at 10.000 x g for 10 min at 4°C
- Remove the supernatant
- Wash the pellet with 500 µl of 75% ethanol
- Centrifuge samples at 10.000 x g for 2 min at 4°C
- Remove the supernatant
- Dry the RNA pellet at least 10-30 min
- Add 60 µL of DEPC-water and incubate at 4°C over night
- Perform DNAse treatment (see ThermoFisher_DNAseI protocol)
- Store RNA at -80°C
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