large scale T7 in vitro transcription (1 mL)

1°) Use autoclaved water that can in addition be treated with Diethyl pirocarbonate to remove pyrogens and potential RNases:

Add 0.1% DEPC (vol./vol.) in the water and incubate overnight at 37°C, with a rotating magnetic stirrer. Autoclave water . (DEPC, Sigma D 5758)

2°) Prepare 10X buffer: 400 mM Tris-HCl pH8.1

10 mM spermidine

3°) Assemble the reaction described below respecting the same order:

422 µL H2O

100 µL 10X buffer

80 µL ATP 50 mM

80 µL CTP 50 mM

80 µL GTP 50 mM

80 µL UTP 50 mM

28 µL MgOAcetate 1M

100 µL DTT 50 mM

10 µL Triton X100 1%

10 µL DNA template* 5 µM

10 µL T7 RNA pol. (home made but follow manufacturer instructions for commercial enzyme)

*: the type of template can vary here: linearized vector, annealed oligos

4°) Incubate 5 hrs at 37°C

if reaction works, the reaction mixture should turn with turbid due to accumulation of PPi, can start 30-60 minutes after reaction started.

5°) Treat with DNase 1 (determine the number of units needed according to the amount of template used in the reaction and to the manufacturer's suggestion). The enzyme should work in the transcription buffer.

6°) Denature proteins, volume to volume, with phenol buffered with 0.1M citrate pH4.3 (Sigma P 4682) (i.e. add 1mL to the transcription reaction).

7°) Extract with chloroform, volume to volume.

8°) Transfer aqueous phase to a high centrifugation speed tube (i.e. COREX if available) and precipitate overnight 

with parafilm to close the tube:

1 mL Transcription reaction

111 µL NaOAcetate 3M pH5.2

3.350 mL absolute EtOH (precooled at -20°C)

9°) centrifuge 30 minutes , 10 000xG at 4°C

10°) the pellet obtained should contain both the RNA and the PPi that will make it look solid, redissolve it in 0.5M EDTA pH8.0 , 50 µL should be sufficient here. Don't shake the redissolving pellet on a vortex, but prefer to leave it dissolving at room temperature for a while without shaking. Handshake the tube to control dissolution. Partial dissolution of the pellet is not a problem since RNA should redissolve in water anyway.

acknoledgements: Camille Mary