Griffitts:Reverse transcription: Difference between revisions
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* Place in (48–52°C) water bath for 60 minutes | * Place in (48–52°C) water bath for 60 minutes | ||
* Store in -20°C freezer | * Store in -20°C freezer | ||
When ready proceed to [[Griffitts:TaqPCR|standard PCR]]. | When ready proceed to [[Griffitts:5'RACE|5'RACE]] or [[Griffitts:TaqPCR|standard PCR]]. Note: For [[Griffitts:TaqPCR|standard PCR]] use 1 μL of RT reaction in each PCR. | ||
==Reaction recipe== | ==Reaction recipe== |
Latest revision as of 11:04, 11 April 2008
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
- 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