Biomedical Engineering Reference
In-Depth Information
Fig. 5.4.
Upward displacement gradient unloader
wall, or locate the tube vertically, fix the outlet tubing of the gradient
mixer to the bottom of the centrifuge tube, and start the gradient
with Soln. B. Store the filled tubes in a refrigerator overnight (avoid
any vibrations).
Cover the sucrose gradient with 0.10 ml RNA-containing sample
and precisely tare the tubes which will be opposite in the rotor.
Spin the samples with 200 000
g
max
at 4
◦
C for 15 - 17 h.After
the run, displace the gradient using a more dense solution, e.g., 40%
sucrose in Soln. A, colored by a droplet Amido Black 10 B solution.
The principle of a displacement apparatus is shown in Fig. 5.4. The
RNA content of the fractions is measured either by reading the UV
absorption at 260 nm or, if labeled material was used, by counting
the radioactivity. To monitor the sucrose gradient, estimate the
refractive index of the obtained fractions (concentration, density
and refractive index of sucrose solutions are given in Table 8.17).
×
5.3.4 Denaturating RNA Gradient Centrifugation
Amix1ml of 1 M HEPES with 99 ml of high-quality DMSO
4
Solutions/Reagents
B
2.5% sucrose (w/v) in Soln. A
C
5.0% sucrose (w/v) in Soln. A
D
7.5% sucrose (w/v) in Soln. A
E
10.0% sucrose (w/v) in Soln. A
DMSO (ultra pure)
DMF (ultra pure)
4
Distil DMSO in a nitrogen atmosphere; b.p.
18
86
◦
C,b.p.
8
63
◦
C,n
2
D
1.4787
