Griffitts:Reverse transcription

Procedure
When ready proceed to 5'RACE or standard PCR. Note: For standard PCR use 1 μL of RT reaction in each PCR.
 * Prepare the reaction recipe
 * Place in 65°C water bath for 5 minutes
 * Place on ice for 1 minute
 * Centrifuge for 20 seconds
 * Add 8 μL 5X 1st strand buffer
 * Add 2 μL 0.1 M 1 M DTT (from -20°C stock)
 * Add 2 μL Superscript III RT (in freezer) to each reaction
 * Add 2 μL dH2O to each control
 * Place in (48–52°C) water bath for 60 minutes
 * Store in -20°C freezer

Reaction recipe

 * 12 μL dH2O
 * 4 μL Primer (1:20 dilution)
 * 10 μL RNA from 50-μL prep
 * 2 μL RNAse-free 10 mM dNTP mix

Primer Dilution
Note: This makes a 1:20 dilution of your 100 μM gene-specific primer
 * 38 μL of RNAse-free dH2O)
 * 2 μL primer