RT-PCR using Superscript III Part I: First-Strand cDNA Synthesis Note: remember to spin down reagent tubes as they may contain small volumes! Also note that many of these reagents have similar names (for example, RNA, RNAseOUT, RNAseH, or DTT, Oligo(dT), dNTP, etc.). Pay attention and don’t mix them up! 1. Combine the following in a 1.5ml microcentrifuge tube: 8μl RNA (your RNA, or borrow from a friend; dilute if over 1μg / μl) 1μl 50μM oligo(dT)20 1μl 10mM dNTP mix 2. Denature the RNA by incubating at 65°C in a water bath for 5 minutes 3. Place on ice for at least 1 minute to anneal the oligo(dT) to the poly(A) tail of the mRNAs 4. Prepare cDNA synthesis mix in a new tube 2μl 10x RT buffer 4μl 25mM MgCl2 2μl 0.1 M DTT 1μl RNaseOUT 1μl SuperScript III RT 5. Add 10μl of cDNA Synthesis Mix to the RNA/primer mix, mix gently, centrifuge briefly 6. Reverse translate the RNA to DNA by incubating for 50 minutes at 50°C 7. Terminate the reaction by incubating at 85°C for 5 minutes 8. Chill on ice to cool 9. Centrifuge briefly. Add 1μl RNase H. 10. Remove RNA by incubating for 20 minutes at 37°C Part II: Amplification of target DNA 1. 2. Combine the following in a PCR tube 2μl cDNA (the tube from part 1, end of Step 10) 10μl 2X Brilliant QPCR Master Mix with low ROX 1μl primer mix (contains both forward and reverse) 1μl SYBR green 1 (already diluted 1/1000) 6μl H20 Spin down and hand in to TA to amplify and quantify cDNA in a real-time PCR instrument