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RESEARCH DIVISION Laboratory Manual

 


 

Primer Labelling for Primer Extension Aassay and Primer Extension

Mix in a 20ul final volume:

2ul 10x kinase buffer
1ul oligonucleotide primer (5pmoles/ul)
200uCi 32P-gATP (crude, ICN, 7000Ci/mmole)
10 U polynucleotide kinase
ddH2O to 20ul

Incubate at 37C for 30-45 minutes. Heat at 65C 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:
500mM Tris pH 8.0
100 mM MgCl2
10mM spermidine
1 mM EDTA
50mM DTT (add fresh before using)

All solutions, eppendorfs and pipette tips need to be RNAase-free and sterile. Use gloves and work

carefully.

  1. Precipitate the RNA with 5' end-labelled primer in 0.3M NaOAc with 2.5 volumes of ethanol. If working with low amounts of RNA add some yeast RNA carrier (approx. 10ug). Wash pellet with 70% ethanol and dry.
  2. Resuspend RNA in 8 ul ddH2O and add 2 ul of 5X annealing buffer (1.25M KCl, 10mM Tris-HCl pH 7.9, 1mM EDTA). Double this volume if more than 20 ug of RNA are used, ie., use 16 ul ddH2O and 4 ul of 5X buffer.
  3. Incubate at 60 for 90 minutes. Spin briefly every 30 minutes to avoid drying of the RNA. The annealing temperature depends on the length and GC content of the primer. The above is O.K. for 20-30 base primers with 50-70% GC content.
  4. Add 23 ul of PE mix (10mM MgCl2, 5mM DTT, 100 ug/ml actinomycin D, 0.33mM dNTP's, 20mM Tris pH 8.7 at room temp., pH 8.3 at 37C, store at -20C in the dark) and 10 U of reverse transcriptase (Life Sciences). Mix carefully, spin briefly, mix again. (Use twice these quantities if annealing was done in 20 ul.)
  5. Incubate at 37C for 1 hour.
  6. Add 0.3 ml chilled ethanol. Put in dry ice bath for 15 min. Spin at 4C for 10 min. Wash in 70% ethanol. Dry.
  7. Dissolve in 2 ul of 0.1N NaOH, then add 4 ul of formamide dyes. Boil for 2 min., chill on ice and load on sequencing gel.
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