20.109(S10):Prepare RNA by IVT (Day3)
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Revision as of 19:54, 5 January 2010 by AgiStachowiak (Talk)
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Contents
Introduction
Protocols
Part 1: In vitro transcription
The table below lists the amount of each reaction component needed for an 80 μL IVT. First, you should calculate how much total IVT Master Mix to make (2 or 4 rxns depending on whether you and your partner share one mix or not, plus 10% excess) and check your calculations with the teaching faculty if desired. Next, you can aliquot the appropriate amount of Master Mix into a number of eppendorf tubes, then add the relevant DNA to each labeled tube.
| Reagent | Amount for 1 reaction (μL) | Amount for N reactions + 10% |
|---|---|---|
| G7 buffer (2.5X stock) | 32 | |
| 1N KOH | 4.48 | |
| Pyrophosphatase | 4 | |
| NTPs | 22.4 | |
| T7 polymerase | 4 | |
| DNA | 13.1 | N/A |
Place your reaction tubes on the 37 °C heat block and write the time in your notebook. When everyone is ready, we will begin the journal article discussion. Also have Atissa come in and give her presentation talk?
