|Random Hexamer Labelling of cDNA Probes |
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.
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
|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: email@example.com |
David Bowtell PMCI October 1998