NOTE: The 5X oligo-labelling buffer (OLB) is made in large batches and stored at -20°C for at least a year. If you do this, keep it in aliquots of a size such that you thaw and refreeze a particular tube for not more than a month or two - after this it may become less effective. For all normal uses it is only necessary to use one hot nucleotide (I use 32P-dATP). The OLB has the necessary cold nucleotides, so you need to make another one if you want to use two hot ones.

The OLB is made from several component solutions.

Solution O:

Solution A:

Solution B:

Random hexamers (#27-2166.01-Pharmacia), 90 OD units Units/ml in TE. One OD260 equals an optical density of 1 in a 1cm path cuvette. This corresponds to about 33ug of oligonucleotide.

1.0 ml 5X OLB

DNA Labelling

1. Boil DNA (100ng - max) in 32ul (made up with water) for 5 minutes.
2. Quench on ice. Set up the reaction as follows:

   -	DNA in 32ul (DNA and DDW as above)
   -	10ul 5X OLB-A
   -	2ul 10mg/ml BSA (optional)
   -	5ul a-32P-dATP (3000 Cl/mmol)
   -	0.5-1.0ul (2-5U) Klenow

The reaction is done at room temperature; most incorporation has occurred by one hour, but it continues to increase till about 3-4 hours. Unlike nick translation, it does not then diminish, and if it's convenient, it's fine to leave it overnight.

3. Separate unincorporated nucleotides by your favourite method: an easy one is 50ul 4M NH4Acetate, 200ul ethanol and 1ul 10mg/ml tRNA. Vortex (DON'T freeze) and spin for 5'. Resuspend pellet in 200ul. 1ul against probe on our (HFI) mini monitors gives 200-300 cps.

Incorporation should be between 50% and 80%, unless you used less than 20-30ng of DNA. The specific activity can reach about 3x109 dpm/ug.