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RESEARCH DIVISION Laboratory Manual

 


 

Rapid Amplification of mRNA Ends By PCR (RACE)

Buffers

10X PCR buffer

100 mM Tris.Cl (pH 8.3)
500 mM KCl
15 mM MgCl2

10X tailing buffer

1 M potassium cacodylate
250 mM Tris.Cl (pH 7.6)
10 mM CoCl2
2 mM DTT
  1. 3'-end Amplification of mRNA by the PCR
    1. Synthesize the first strands of cDNAs using a (dT)17-adaptor primer.
      - Dissolve 1-5 ug of poly (A)+ RNA or 10-20 ug of total RNA in 10 ul of water (DEPC treated and autoclaved beforehand). Heat at 65C for 5', chill on ice.
      - Add 2 ul of 10X PCR buffer, 2 ul of dNTP's (each 10 mM), 2 ul of DTT (10 mM), 0.5 ul of RNasin, 3 ul of (dT)17-adaptor (100ng/ul) and 1 ul of AMV reverse transcriptase.
      - Incubate the reaction at 42C for 1-2 hr, then heat at 95C for 5'. Dilute the reaction mixture with 180 ul of TE (cDNA poll).
    2. Amplification of the target cDNA.
      - 1-10 ul aliquots from above cDNA poll, add 10 ul of 10X PCR buffer, 4 ul of adaptor (50 ng/ul), 4 ul of gene-specific primer (50 ng/ul), 10 ul of dNTP's (2 mM each), water to 100 ul. Then add 0.5 ul of Taq polymerase.
      - Do 40 cycles (cycle conditions depend on primer, size of the PCR product etc.)
      - Load 10 ul aliquots of PCR products plus 5 ul of sucrose loading dye on a agarose gel.
      - Southern blot analysis can be further performed using a internal gene-specific probe.
  2. 5'-end Amplification of mRNA by the Nested PCR
    1. Synthesize the first strand of cDNA using a gene-specific primer 1.
      - Reverse transcribe RNA as above, but substitute 3 ul of gene-specific primer 1 (50 ng/ul) for (dT)17- adaptor.
    2. Remove excess primer 1 by a centricon-100 spin filter
      - After heating the RT mixture at 95C for 5', dilute with 2 ml of water, and transfer to a centricon-100, centrifuge at 3.5K (SW34 rotor) for 45'. Repeat and collect retained liquid.
    3. Tail the 3' end of the target cDNA
      - Heat 15 ul aliquots of above first-strand cDNA at 65C for 5', chill on ice.
      - Add 2 ul of 10X tailing buffer, 2 ul of 10 mM dGTP (or dATP), 1 ul of terminal transferase.
      - Incubate the reaction mixture at 37C for 10-15'.
      - Heat at 65C for 10'. Then add 200 ul of TE, extract by phenol/chloroform.
      - Dilute the mixture with 2 ml water. Transfer to a centricon-100 spin filter and Centrifuge at 3.5 krpm for 45'.
    4. Amplification of the tailed cDNA by the PCR.
      - Do the PCR as above, using 10 ul of the tailed cDNA, 4 ul of gene-specific primer 2 (50 ng/ul), and 4 ul of (dC)15-adaptor (if the template was tailed by dGTP) or 4 ul of (dT)17-adaptor and 4 ul of adaptor (if the template was tailed by dATP). Primers concentration is 50 ng/ul.

NOTES:

  1. Primers:

- (dC)15-adaptor(#1504): 5'>AAA AGA TCT GTC GAC CCC CCC CCC CCC CC<3'
- (dT)17-adaptor(#1394): 5'>GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT<3'
  1. In the nested PCR, gene-specific primer 2 should located in the first-strand cDNA.

Reference:

Frohman, M.A., Dush, M.K., & Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA 85, 8998-9002.

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