Biomedical Engineering Reference
In-Depth Information
Fill 1/5 of the effective volume of a centrifuge tube with Soln. B,
cover it with the same volume of Soln. C, followed by Soln. D and
Soln. E. Store the centrifuge tubes vibrationless at RT overnight.
A linear gradient is formed by diffusion during this time.
Mix 60
µ
µ
µ
l of RNA solution with 275
l DMSO, followed by 165
l
µ
DMF. Cover each gradient with 200
l of this RNA solution.
g
max
at 25
◦
C for 15 - 17 h.
RNA monitoring by UV reading is not possible, because DMSO
absorbs below 275 nm, i.e., within the range of the nucleic acid
absorption.
Spin the samples with 200 000
×
References
Rickwood D (ed.) (1992) Preparative centrifugation: a practical approach.
Oxford University Press, London
5.3.5 Isopycnic Centrifugation
Isopycnic (buoyant density, equilibrium) separations should be
performed in fixed angle or vertical rotors, because swing-out ro-
tors give relatively large differences in centrifugal fields, i.e. the
difference between g
min
and g
max
is much larger than in fixed angle
rotors.Ifavailable,verticalrotorsshouldbepreferred,becausethey
give shorter separation times than fixed angle rotors if run at high
speed.
If self-forming cesium salt gradients are used, take care to be
within the solubility of the salt at the given temperature to avoid
crystallizations during centrifugation, since salt crystals may de-
stroy the rotor during run (solubilities of cesium chloride and
sulfate are given in Table 5.4).
The maximal speed of a rotor has to be reduced if solutions
with densities
ρ
/
ml are used. The proper revolutions per
minute N
red
are calculated by
>
1.2 g
1.2
ρ
=
N
red
N
max
·
5.3.5.1 Purification of High Molecular Weight DNA
in CsCl Gradients
This protocol is an example of separation using a self-generating
density gradient.
A 0mM Tr i s
·
HCl, 5 mM EDTA, pH 7.8
Solutions/Reagents
cesium chloride (ultrapure)
Dissolve 2.0 g cesium chloride (ultrapure) in 2.0 ml of Soln. A,
containing 5 - 8
g DNA. Stir with a glass rod at 20
◦
C until the
salt is completely dissolved. Pour the solution into a centrifuge
µ
