CDNA synthesis

Refer to section on troubleshooting

24. Prepare the following master mix, accounting for 1-2 additional reactions per gene.

Ingredient

Per reaction

For x reactions

5X SuperScript II buffer

10 pl

x x 10 pl

10 mM dNTPs

5 pl

x x 5 pl

0.1 M DTT

5 pl

x x 5 pl

BSA (RNase-free, optional)

1.25 pl

x x 1.25 pl

Random primers

0.25 pl

x x 0.25 pl

RNA guard

1.25 pl

x x 1.25 pl

Superscript II reverse transcriptase

1.25 pl

x x 1.25 pl

Molecular biology water

1 pl

x x 1 pl

Total

25 pl

x x 25 pl

25. Using the LTS-200 repeating pipette, add 25 pl of the reverse transcription master mix to 200 pl PCR strip tubes labeled with the date and sample number on the side.

26. Add 25 pl of the DNase-treated RNA to each tube. This will contain 1 pg of RNA.

27. Incubate at the following temperatures using a PCR thermal cycler: 26°C for 10 min (to allow the random hexamers to anneal), 42°C for 45 min (reverse transcription) and 75 °C for 10 min (to inactivate the reverse transcriptase).

28. The resulting cDNA may be analyzed immediately by real-time PCR or stored at -20°C or -80°C.

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