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RESEARCH DIVISION Laboratory Manual

 


 

Random Hexamer Labelling of cDNA Probes

NOTE: The 5X oligo-labelling buffer (OLB) is made in large batches and stored at -20C 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:

- 1.25M Tris HCl, pH8.0
- 1.25M MgCl2

Solution A:

- 2mls Solution O (for OLB - A)
- 36ul 2-mercaptoethanol
- 10ul 100mM dCTP (no C but add A for OLB-C).
- 10ul 100mM dGTP
- 10ul 100mM dTTP

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

- 0.2mls Solution A
- 0.5mls 2M HEPES, pH6.6
- 0.3mls Solution B

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.

  1. 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.

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998