Please read the excellent descriptions of handling RNA and of cDNA synthesis by J. Sambrook, E. F. Fritsch, and T.Maniatis in Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Second Edition, New York, 1989). The methodology for the synthesis of the first strand of the cDNA summarized below is based primarily on this source.

1st Strand cDNA Synthesis
     Dry in a vacuum desiccator:
          5 µg poly-A RNA
          50 µCi (5 µl) alpha-32-P-dCTP

     Add to the RNA:
          5 µl H₂0
          0.5 µl 100mM Me(Hg)OH [in fume hood, DANGEROUS]
        
     Incubate at room temperature for 10 min. Then add:
          1 µl 700mM B-mercaptoethanol (BME) diluted in H₂O
          0.5 µl RNAsin (15units; stored at -30°C)

     Incubate at room temperature for 5 min. Then add:
          5 µl oligo-dT (@1mg/ml)
          2.5 µl 1M Tris-HCl, pH 8.3 (pH at 42°C)
         
          2 µl reverse transcriptase @ 19U/µl (higher than Maniatis)

     Incubate at 42°C, 3 hr. Add 1µl of 0.5 M EDTA to stop the reaction. Extract with 15µl phenol + 15µl CHCl₃.

Determine the efficiency of first strand cDNA synthesis:
    Count:

1. 1µl of the reaction mixture spotted on a GF/C filter. This will measure the total radioactivity in the sample.
   
2. 1µl of the reaction mixture plus 5 µl of a 40 mg/ml yeast tRNA stock (dissolved in water) and 5 ml of 10% TCA. Incubate on ice, 15 min. Filter through a GF/C filter. Wash the filter 4 times with 5 ml of cold 10% TCA, followed by 5 ml of 95% ethanol. This will measure the radioactivity incorporated into high molecular weight nucleic acids.

    Use the following to calculate the total µg of cDNA.

1. Moles of dCTP = "a" =
   (% incorporated above)(molar concentration of dCTP)
   where the molar concentration = cold dCTP concentration since ³²P-labeled dCTP is just a chase).
   
   
2. Labeled moles expected with 100% efficiency = b =
   (about 25% of each base)(µg RNA)/(about 330 g/mole)
   
   
3. % Efficiency = a/b x 100

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