Griffitts:Reverse transcription

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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 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

When ready proceed to 5'RACE or standard PCR. Note: For standard PCR use 1 μL of RT reaction in each PCR.

Reaction recipe

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

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