ISOLATION OF L. tarentolae KINETOPLAST-MITOCHONDRIAL FRACTION UNDER ISOTONIC CONDITIONS

1. Grow Lt in BHI medium. Harvest after 48 hour at 100-150 x 106 /ml (late log phase) in 500-ml plastic centrifuge bottles. 5000 rpm, 10 min, 4C. Wash cells in cold SHE. 5000 rpm, 10 min.
2. Resuspend cells in SHE at 1.2 X 109 cells/ml. Disrupt in the Stansted Power Fluid apparatus at 60-72 psi. Cool the collection funnel at 5C. This results in breakage of >90% of the Lt. You can pass through a second or even a third time to rupture all the cells.
3. 11,000 rpm, 15 min, 4C (Sorvall GSA rotor; 19600 XG). Decant, avoiding the loose pellet. Resuspend well in SHM (5 ml per each 30 ml of lysate). Add 3 mM MgCl2. Add 1/200 stock DNase I. Digest in ice bath for 1 hr. Add an equal volume of cold SHE. 11000 rpm, 15 min, 4C (Sorvall GSA rotor; 19600XG). Aspirate off supernate. Pellet should be well packed.
4. Resuspend well in a 2X volume of SHE. Add 5 ml 76% RSTE per liter of original L. tarentolae culture volume. Vortex and let sit on ice for 5 min to remove air bubbles. Layer 6 ml under 32 ml of 20-35% RSTE gradient using a syringe/needle/tubing assembly. Step gradient is poured and stored frozen. Remove several hr before use for thawing at 4C to create linear gradient. 24000 rpm, 2 hr, 5C, SW28 rotor.
5. Tap gradient from side with syringe to recover kinetoplast band. Do not collect the upper part of the band as it contains intact cells and cell ghosts. Pool in a 250-ml centrifuge bottle and fill with SHE. 11000 rpm, 15 min, 4C, Sorvall GSA rotor, 19600 X G, or SS-34 rotor, 11500 rpm, 15 min, 4C. Recover pellets. Resuspend in SHE. Wash once in SHE.
6. For K-RNA isolation, resuspend the pellets in 10 mM Tris/HC1 pH 7.4. Shell freeze in dry ice/ethanol. Store at -20C.
7. For in vitro editing assays resuspend in 10% glycerol, 20 mM KCl, 20 mM HEPES (pH 7.5), 0.2 mM EDTA.

Solutions:

SHM: 0.6 M sorbitol (can also use 0.25 M), 20 mM Hepes, 2mM MgCl2, pH 7.5 (for 2 l: 91.1 g sorbitol, 80 ml 0.5M Hepes, 20 ml 0.2M MgCl2, adjust to pH 7.5 with HC1 or NaOH).
SHE: 0.6 M sorbitol (can also use 0.25 M), 20 mM Hepes, 2mM EDTA, pH 7.5 (for 2 l: 91.1 g sorbitol, 80 ml 0.5M Hepes, 20 ml 0.2M EDTA, adjust to pH 7.5 with HC1 or NaOH).
76% RSE: dissolve 8.556 g sucrose and add 50 ul 0.2M EDTA to 100 ml 76% Renografin (commercially available at 76%). Store frozen.
20% RSTE: 26.3 ml of 76% Renografin. Add 8.56 g sucrose with 10 ml 0.2M Tris HC1 pH 7.9 and 50 ul 0.2M EDTA Bring vol to 100 ml. with water. Adjust density to 1.14 - 1.15 g/ml (nD at 25C=1.3736).
35% RSTE: dissolve 8.55 g sucrose and add 10 ml 0.2M Tris HC1 pH 7.9 plus 50 ul 0.2M EDTA to 46.1 ml of 76% Renografin. Bring vol to 100 ml. with water. Adjust density to 1.26 g/ml (nD at 25C=1.3972).
DNase I (Sigma) electrophoretically pure: 2 mg/ml in 10 mM Tris/HCl pH 7.4, 10 mM CaCl2, 50% glycerol. Store at -20C.
Renografin gradients: 15 ml 35% RSTE in SW28 tubes. Layer 15 ml 20% RSTE on top and freeze at -20C. Thaw at 4C a few hr before use. Layer sample at the bottom of the tube using a 12 ml syringe with #18G needle and polyethylene tubing.
0.2M EDTA: 74.45 g Na3 EDTA/l, adjust pH to 7.9 with NaOH.