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Contributor: Suprya Jayadev
Date: August 27, 1991
1) Add 22 ml of light Percoll to a centrifuge tube.
2) Using a 20 G 3 1/2 in spinal needle on a 10 cc syringe, carefully underlay
with 6 ml of dense Percoll.
3) Centrifuge tubes at 48,000xg (~20,000 rpm in JA-20 rotor in Beckman J2-21
high speed centrifuge) for 15 mins, 40C.
4) Carefully layer supernatant on top of the preformed gradient; without
disrupting the gradient!!
--> In addition to gradient containing sample, should also run a marker
gradient loaded with colored marker beads of known density (Pharmacia).
5) Centrifuge samples at 48,000xg , 40C, 15 mins, as before.
6) Collect 1 or 2 ml fractions at 40C by aspiration from the bottom of the
gradients through a pipet tip attached to polyethylene tubing and peristaltic
7) Should have collected 19 or 38 fractions into 5/8 X 3 in,10.4 ml capacity
centrifuge bottles (Beckman # 355603) during the previous step.
--> Determine the distances of bead migration in the marker gradient.
8) Weight balance tubes using Percoll balancing solution.
9) Centrifuge fractions at 180,000xg (~60,000 rpm using the 70.1 Ti rotor
in the Beckman L7-65 ultracentrifuge) for 30 minutes.
--> or at 100,000xg for 90 minutes
10) Collect supernatants and discard pellets.
Relaxation buffer (without EGTA):
100 mM KCl
3 mM NaCl
1 mM ATP(Na)2
3. 5 mM MgCl2
10 mM PIPES pH 7.3
Light Percoll (final density 1.040 g/ml)
10 ml 10X relaxation buffer with EGTA
26.4 ml undiluted Percoll (density 1.130)
63.6 ml dH2O
--> relaxation buffer makes Percoll isotonic
--> relaxation buffer should contain 12.5 mmol/l EGTA
Dense Percoll (final density 1.120 g/ml)
3 ml 10X relaxation buffer with EGTA
26.4 ml undiluted Percoll
0.60 ml dH2O
Percoll balancing solution (final density 1.122 g/ml)
5 ml 10X relaxation buffer with EGTA
45 ml undiluted Percoll