6. Wash the filter discs six times with 2 ml 10% TCA solution and
dry the filter discs thoroughly, for example using an infra-red
lamp. Avoid overheating and possible charring of the discs.
7. Count the dried filter discs by liquid scintillation or Cerenkov (32P)
and count with the samples set aside in step 3.
8. Determine % incorporation and probe specific activity as in
section A6.
6.3. Appendix III. Removal of unincorporated nucleotides
Removal of unincorporated nucleotides is sometimes desirable to
reduce background produced by the probe during hybridization. It is
considered important to remove these free nucleotides particularly if
the radioactive probe is to be kept for several days before use or the
incorporation is less than 50%. If 32P or 33P-labelled probes are to
be used in combination with GE Healthcare’s new Rapid-hyb buffer
(RPN1635/6), purification is not required unless the probe is to be
used more than 24 hours after preparation. Probes can be purified
by Sephadex chromatography or selective precipitation (8,9).
A. Sephadex™G-50 spin columns
Probe reaction are passed through columns packed with Sephadex
G-50, which retains the free nucleotides within the column matrix.
A number of pre-packed columns are commercially available.
However columns may also be prepared as indicated below:
1. Equilibrate Sephadex G-50 in TE buffer either overnight or at 65°C
for 1–2 hours.
2. Plug a 1.0 ml syringe with a piece of siliconized glass wool.
3. Fill the syringe with the equilibrated Sephadex. Place in a 15 ml
conical tube, in which a decapped 1.5 ml microcentrifuge tube
has been inserted. Centrifuge at 1600 g for 5 minutes. Remove
32