Griffitts:Reverse transcription

Procedure

• 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 1^st 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

When ready proceed to standard PCR. Use 1 μL of RT reaction in each PCR. Then proceed to 5'RACE

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

• 38 μL of RNAse-free dH₂O)
• 2 μL primer

Note: This makes a 1:20 dilution of your 100 μM gene-specific primer