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For purification of DNA fragments generated by PCR, the 100 µL reactions were
diluted with 2.0 mL of TE Buffer (10 mM Tris-HCL, pH 8.0, 1 mM EDTA) and
centrifuged at 5,000 × g for 15 minutes. The sample reservoir was filled with
another 2.0 mL of TE buffer and centrifuged again as above. The purified PCR
products were collected by a reverse spin and the recovered DNA quantified by
a fluorometric assay.
Biological Activity
In lab tests, 1 mL of lactic dehydrogenase (type 5S rabbit muscle,
M4 isoenzyme, 99% homogenous) containing 5 units of enzyme
activity and 1 mg/mL bovine serum albumin (to minimize
nonspecific adsorption) was spun in a Centricon-30 device for 30
minutes at 2000 × g. All five units of the original activity were re-
covered with 90% recovery of bovine serum albumin.
[A unit = amount of activity to reduce 1 µmole of pyruvate to lactate
per minute at pH 7.5, 37 °C.]
0%
20%
40%
60%
80%
100%
137
301
657
1159
PCR Size, (bp)
Re
c
ov
e
ry
,
(
%
of
Cha
lle
nge
)
Purification of PCR Fragments Using Centricon Device,
50,000 NMWL
Summary :
[A unit = amount of activity to reduce 1 µmole of pyruvate to lactate per minute at pH 7.5, 37 °C.] 0% 20% 40% 60% 80% 100% 137 301 657 1159 PCR Size, (bp) Re c ov e ry , ( % of Cha lle nge ) Purification of PCR Fragments Using Centricon Device, 50,000 NMWL
Tags :
pcr,actiity,boine,buffer,fragments,purification,100,albumin,units,dna,centrifuged,serum,minutes