diff --git a/assays/RNA_extraction/protocols/ThermoFisher_DNAseI.pdf b/assays/RNA_extraction/protocols/ThermoFisher_DNAseI.pdf new file mode 100644 index 0000000000000000000000000000000000000000..f15bea933fb4964eca2d1143409b670d13241932 Binary files /dev/null and b/assays/RNA_extraction/protocols/ThermoFisher_DNAseI.pdf differ diff --git a/assays/RNA_extraction/protocols/trizol_RNA_extraction.txt b/assays/RNA_extraction/protocols/trizol_RNA_extraction.txt new file mode 100644 index 0000000000000000000000000000000000000000..d80fb95dc1c55354483ac232bd4fa66d923f3c29 --- /dev/null +++ b/assays/RNA_extraction/protocols/trizol_RNA_extraction.txt @@ -0,0 +1,26 @@ +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