HELP!How do you properly count radiolabelled DNA on filter paper disks?
patrick.walter at bc.biol.ethz.ch
Thu Jun 15 10:46:03 EST 1995
I am separating radiolabelled denatured mtDNA strands on
CsCl density gradients (more details below). Then I
determine the amount of radioacvity in fractions from
the gradient by using the technique described by Bollum (Bollum, F.J.,
Filter Disk Techniques for Assaying Radioactive Macromolecules,
in: Procedures in nucleic acid research, ed: Cantoni, G.L. and
Davies, D.R., Harper and row, New York (1966)).
Somewhere in this technique I am making a slight error (I
believe it is the final drying time, I now use a min of 15 min),
because something changes the max CPM I observe by 3
orders of magnitude (for equal radioactive incorporation).
Does anyone have any suggestions? Any comments would be
I am now incorporating radiolabeled nucleotides into mtDNA
during incubations of intact mitochondria in vitro. The
mtDNA is then purified and separated from unincorporated
nucleotides. Then the heavy and light strands of mtDNA are
separated by, first heating at 100°C (in the presence of DMSO
to keep from renaturing) for 10 minutes, then separating the
strands during density gradient centrifugation using 1.7 g/ml
of CsCl. Fractions from these gradients (40/gradient) are then
collected by bottom puncture and their radioactivites
determined by spotting on GF/C filters using the method of
Bollum (see above for reference)
Basic Procedure that we use:
1. Use Whatman GF/C glass microfiber filters (cat. No. 1822 024) 2.4 cm dia.
2. Soak filters in 0.1% Na4P2O7.10H2O.
3. Allow to dry for 15 min (the filters are not completely dry in this time).
4. Apply 45ul of radioactive DNA sample to each filter.
5. Allow to soak into filter for 5 min.
6. Remove acid soluble materials by immersing filter in ice cold 5% TCA for 10 sec.
7. Wash 2 times in ice cold 5% TCA (5 min. each).
8. Wash 1 or 2 times in ice cold 95% ethanol.
9. Oven dry at 37°C for 15 to 60 min.
10. Place filter in scintillation cocktail (Lumasafe, Lumac LSC), and analyse in a
suitable liquid scintillation counter.
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