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any liquid from the microcentrifuge tube. Refill with Sephadex
and centrifuge as before. Continue until the column is packed to a
volume of 1 ml.
™ Sephadex is a trademark of GE Healthcare
4. Add a volume of TE buffer equal to the reaction volume, to the
top of the column and centrifuge, as in step 3. A minimum of 50 µl
should be applied to the column.
5. Repeat once more to ensure fractions of the correct size are
collected from the column.
6. Place the column in a clean 15 ml conical tube containing a
decapped 1.5 ml microcentrifuge tube.
7. Apply the DNA sample to the column. Centrifuge as before. The
purified probe is collected in the microcentrifuge tube.
B. Selective precipitation of labelled DNA
The following protocol leads to precipitation of DNA greater than
about 20 nucleotides in length with unicorporated nucleotides
remaining in solution. Recovery of the labelled DNA by this method
varies according to the DNA concentration and size, and may be as
low as 50%.
1. Add one volume of 4 M ammonium acetate, pH4.5 to the nick
translation reaction, and mix gently by pipetting up and down.
2. Add four volumes of ethanol, mix by inversion. Chill the mixture for
15 minutes in a dry-ice ethanol bath or place at -70°C for at least
30 minutes.
3. Thaw the mixture if necessary by placing at 37°C for 2 minutes.
4. Spin in a microcentrifuge for 15 minutes. Carefully aspirate and
dispose of supernatant in a suitable manner.
5. Wash the pellet once in 0.5 ml of 0.67 M ammonium acetate,
pH 4.5, 67% ethanol at room temperature by gentle inversion,
centrifugation and aspiration.
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