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1st Strand cDNA synthesis 1. Add chilled sterile dH2O to sample for cDNA synthesis to bring volume to 17 ul total. Microfuge briefly to ensure that all of the sample is at the bottom of the tube. Heat in 70ºC water bath for 10 minutes. Immediately transfer to ice and chill for 10 minutes. 2. Add 1 ul 0.25 M DTT 1 ul RNase inhibitor (RNasin) 5 ul 5X reaction buffer, mix sample with pipet tip. Make sure that everything is at the bottom of the tube, and add 1 ul reverse transcriptase. Mix gently. 3. Incubate at 41ºC for 1 hour. Add 75 ul dH2O. Stop reaction by transferring tube to ice. Store cDNA at -20ºC.

Basic PCRto test cDNA 1. Set up a reaction mix containing primers, dNTPs, buffer and Taq enzyme. We will use primers for the 'housekeeping' gene GAPDH.

Stock dH20 F primer R primer 10X buffer dNTPs Taq pol TOTAL

For 1 50 ul rxn 31.5 ul 2 ul 2 ul 5 ul 4 ul 0.5 ul

For20rxns 630 40 40 100 80 10

45 ul

900 ul

2. Set up reactions; bring to 50 ul final volume by addition of dH20 or cDNA. Use thin-walled PCR tubes.