Step 1

Prepare suspension of tissue and add 200 µl to 800 µl of Trizol [Gibco/BRL] in Sarstedt tubes.

Step 2

Leave at ambient (room) temperature for 5 minutes.

Step 3

Add 200 µl of chloroform to each sample, mix thoroughly (vortex) for 10-15 seconds, and leave for 3 minutes.

Step 4

Centrifuge at 13 000 rpm in a bench top microcentrifuge for 15 minutes.

Step 5

During Step 4. label fresh tubes and add 1 µl of glycogen to each.

Step 6

Transfer upper clear layer of each supernatant to a fresh tube containing glycogen and add 500 µl of isopropyl-alcohol (propan-2-OL) to each.

Step 7

Thoroughly mix (vortex) and leave at ambient temperature for 10 minutes.

Step 8

Centrifuge at 13 000 rpm for 10 minutes, drain off supernatant and replace with 1 ml of 70% ethanol.

Step 9

Mix gently (vortex) and centrifuge at 13 000 rpm for 10 minutes, after which there should be a pellet.

Step 10

Carefully remove supernatant, preferably by vacuum suction.

Step 11

Air dry for 1-2 minutes, and hydrate each pellet with 20 µl of MQ (or very pure) water.

Step 12

Mix gently (vortex) and store at -70ºC until required.