Nucleic acids purification

Extraction of Drosophila total RNA

1) Collect ~30 individuals in a 15ml Falcon tube and flash-freeze them in dry ice/Ethanol or liquid nitrogen 2) Add 2 ml of Trizol (Invitrogen, ref:15596018)

and homogenize (ultra turrax or

equivalent) 3) Let stand for 10 minutes at 37°C. Centrifuge 10 minutes at 12.000g (2-8°C) 4) Transfer 1.6ml of supernatant in a clean 2ml Eppendorf safe-lock tube (the pellet contains mainly proteins, chitin, sugars and genomic DNA) 5) Add 400µl Chloroform and vortex 15 seconds 6) Let stand for 2 minutes at RT. Centrifuge for 15 minutes at 12000g 7) Transfer the aqueous phase in a 2 ml Eppendorf safe-lock tube (~1ml should be covered – let enough aqueous phase not to take interface) 8) Add 1 volume Isopropanol and mix by inversion (0.5 vol of Trizol) 9) Let stand for 5 minutes at RT 10) Centrifuge 15 minutes at max speed (2-8°C) 11) Discard all the Isopropanol 12) Resuspend the pellet in 500µl H2O 13) Add 1 volume of Phenol:chloroform:isaoamyl alchool** (25:24:1) (Interchim, ref:973351) and mix by inversion 14) Centrifuge 7 minutes at maximum speed (20°C) 15) Transfer the upper phase in a 2ml tube 16) Add 500µl Chloroform:isaoamyl alchool (24:1) 17) Centrifuge 7 minutes at maximum speed (20°C) 18) Transfer the upper phase in a 2ml tube 19) Add 1.5ml of 96-100% Ethanol and 50µl NaOAc 3M pH5.2 20) Centrifuge 15 minutes at max speed (2-8°C)†† 21) Wash with 1ml of 75% Ethanol‡‡ – centrifuge 5 minutes at max speed and discard ethanol by aspiration and let stand the tube open f 2 minutes at room temperature.

**

adjust pH at 8 is fine- acid phenol prevent DNA extraction but this has been done by the Trizol The pellet should be weakly visible after centrifugation in ethanol. After discarding ethanol, the pellet appears translucide. When dried, the pellet must be transparent and looks like a gel. ‡‡ RNA can stand in 75% Ethanol at -20°C ††

Nucleic Acid Purification 14

Nucleic acids purification 22) - Let stand the open tubes for 2 minutes at RT (Do not over-dry RNA which becomes insoluble). 23) - Dissolve the pellet in H2O by pipetting. Take OD260 and OD280: ratio should be between 1.9 and 2.1 24) Aliquot RNA at 500ng/µl or less depending on the protocol use for reverse transcription. 25) Load approximately 1µg on a 1% agarose gel in formamide loading blue with a control RNA to check RNA integrity.

Nucleic Acid Purification 15