RNA extraction using the 'home - made' Trizol substitute
Prepare the components for the home - made Trizol substitute .
Mix the components and stir at room temperature until completely dissolved ( 30 - 60 minutes ) .
Do not heat the solution .
Store at 4 °C in a glass bottle protected from light .
Add 5 volumes of the home - made Trizol substitute to the sample .
When working in 1.5 mL tubes , for practical reasons , the minimal and maximal volume of the sample is 30 and 200 µl , respectively .
Vortex vigorously for 10 - 30 seconds ( depending on the viscosity and protein and nucleic acid concentration of the sample ) .
Incubate at room temperature for 5 minutes .
Add 1 volume of chloroform ( relative to the original volume of the sample ) .
Mix vigorously 10 - 20 times by inverting the tube .
Do not vortex to avoid breaking DNA .
Incubate at room temperature for 5 minutes .
Spin for 10 - 15 minutes at > 12,000 g and 4 °C ( depending on the viscosity and protein and nucleic acid concentration of the sample ) .
Transfer the upper aqueous phase into a new tube .
Avoid the white DNA precipitate at the interphase .
If the sample volume was 100 µl , the aqueous phase should be ~ 450 µl .
Add 1.1 volumes of isopropanol ( relative to the volume of the aqueous phase ) .
If low RNA content is expected in the sample , prior to isopropanol , add > 10 µg of glycogen to facilitate precipitating nucleic acids and spotting the pellet .
Recommended final concentration of glycogen is ~ 50–150 µg / ml .
Mix well by inverting the tube .
Incubate 30 - 60 minutes at 4 °C or at room temperature .
Spin for 30 - 45 minutes at > 12,000 g and 4 °C .
Discard the supernatant .
Add 1 volume of 70 % ( aq . ) ethanol ( relative to the volume of the aqueous phase ) .
Mix gently .
Ensure that the pellet is submerged in the solution and not sticking to the tube wall .
Incubate 10 minutes at room temperature .
Spin for 10 minutes at > 12,000 g and 4 °C .
Discard the supernatant .
Let the pellet dry at room temperature , so that no traces of 70 % ethanol remain .
Drying under vacuum is not recommended because of overdrying that makes it harder to dissolve the pellet .
Completely dissolve the pellet in RNase - free water .
