Acidic PBCV purification
Acidic PBCV purification

1- Pellet two culture flasks (~350 ml ea) for 4 min at 5K in the F14-6x250y rotor

2- Take Chlorella pellets up in 40 ml MBBM, add 40 ul tetracycline, 35ul purified PBCV. Incubate 16hrs under fluorescent illumination with a 16 hr light/8 hr dark cycle at 25C on an orbital shaker (~95 rpm).

3- To a 100ul aliquot of lysate add 8 ul Triton X-100, centrifuge at low speed to make sure the culture has lysed (chlorophyll should be in supernatant.)

4- To the bulk of the lysate add 10 ml of 0.4M trisodium citrate, pH 4.5 and 10mg of protamine sulfate, mix, incubate 20 min at 37C.

5- Centrifuge 3 min at 9K in the SS34 rotor (RT). Pour supernatant into 1x3.5" polycarbonate tubes, discard pellet.

6- Pellet the supernatant for 25 min at RT in the 50.2Ti rotor (polycarbonate tubes). Discard supernatant.

7- Dissolve pellets in a total of 20 ml K-MOPS, pH 7 buffer. Centrifuge 8K for 2 min in the 50.2Ti rotor.

8- Pour supernatants into fresh polycaronate tubes and pellet 17 min at 25K. Dissolve pellets in about 3ml of K-MOPS buffer.