1        MATERIALS

1.1       Re-suspension buffer (150 mM NaCl, 10 mM EDTA, 50 mM Tris 7.5);

1.2       10 % sarkosyl (L-lauryl sarcosil);

1.3       10 mg/mL RNAse A

1.4       10 mg/mL proteinase K

1.5       Phenol

1.6       Chloroform

1.7       Phenol/chloroform/isoamyl alcohol (25:24:1)

1.8       3M sodium acetate pH7

1.9       100 % ethanol or 100 % isopropanol

2        PROCEDURE

2.1        Extraction

2.1.1      Re-suspend parasite in re-suspension buffer to a final volume of @ 500 mL;

2.1.2      Add 5 mL of 10 % sarkosyl;

2.1.3      Add 5 mL of RNAse A 10 mg/mL and incubate for 1 hour at 37°C;

2.1.4      Add 5 mL of proteinase K 10 mg/mL and incubate overnight at 4°C;

2.1.5      Extract with one volume of phenol, mix by inverting, and centrifuge 5 min at 15,000 g;

2.1.6      Extract the supernatent with one volume of phenol/chloroform/isoamyl alcohol, mix by inverting, and centrifuge 5 min at 15,000 g;

2.1.7      Extract the supernatent with one volume of chloroform, mix by inverting, and centrifuge 5 min at 15,000 g;

2.1.8      Collect the supernatent in a new tube.

2.2       Precipitation

2.2.1      Add 1/10 the volume 3M sodium acetate pH7;

2.2.2      Add 2 volumes of ethanol or 0.8 volume of isopropanol;

2.2.3      Mix and leave at -20°C overnight or leave 10 min at room temperature;

2.2.4      Centrifuge 30 min at 13,000 rpm and 4°C;

2.2.5      Remove the supernatant being carefull not to disturb the pellet, and air dry the pellet;

2.2.6      Re-suspend in 50 mL of ddH₂O;

2.2.7      Read OD at A₂₆₀, store at -20°C.