|Primer Labelling for Primer Extension Aassay and Primer Extension |
Mix in a 20ul final volume:
Incubate at 37°C for 30-45 minutes. Heat at 65°C for 15 minutes.
To remove unincorporated label precipitate as follows:
Add 60ul TE + 320ul 2.5M NH4OAc + 20ug RNA carrier + 650ul 100% ethanol (follow this order of addition; don't add the carrier RNA to the reaction first or some RNA will get labelled). Chill on dry ice for 5-10 minutes. Spin in the cold for 15 minutes. Discard supernatant (very hot!!). Resuspend pellet in 180ul TE and add 20ul 3M NaOAc + 500ul 100% ethanol. Chill on dry ice for 15 minutes, spin in the cold for 10 minutes, wash with 70% ethanol, dry and resuspend in 100ul TE (should be ~200cps/ul on minimonitor).
10x kinase buffer:
All solutions, eppendorfs and pipette tips need to be RNAase-free and sterile. Use gloves and work
|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: firstname.lastname@example.org |
David Bowtell PMCI October 1998