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 65°C 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 42°C for 1-2 hr, then heat at 95°C 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 95°C 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 65°C 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 37°C for 10-15'.
      -	Heat at 65°C 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:

2. 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.