Reagents:

  • ddH2O

  • Phenol, water-saturated and buffered (pH 7.5)

  • Chloroform

  • G25 column

  • Linear acrylamide

  • 3M Sodium Acetate pH 7.5 and pH 5.2

  • Ethanol

  • T7 polymerase with 5x reaction buffer and DTT

  • Nucleosides

  • RQ1 RNase-free DNase

 

Protocol:

 

A. Prepare template

  1. Adjust volume of template PCR or linearized plasmid to 200 µl with ddH2O

  2. Phenol/Chloroform extract 3x with 100 µl P/C pH7.5

  3. Pass through G25 column

  4. Add:

    1. 1 µl linear acrylamide

    2. 30 µl Sodium Acetate pH 7.5

    3. 600 µl 100% Ethanol

  5. Mix, precipitate at -80°C for 15 minutes.

  6. Spin 12 minutes at 14,000 rpm

  7. Wash pellet with 70% Ethanol

  8. Wash pellet with 100% Ethanol

  9. Dry pellet for 10 minutes

  10.  Dissolve pellet in 11 µl ddH2O

  11.  Use 1 µl template to determine concentration.

  12.  Use 1.5 µg template in 10 µl ddH2O for transcription reaction.

 

B. In vitro transcription reaction

  1.  Make reaction mix:

    1. 38 µl ddH2O

    2. 20 µl 5x Buffer

    3. 10 µl 100 mM DTT

    4. 5 µl 10 mM rGTP

    5. 5 µl 10 mM rATP

    6. 5 µl 10 mM rUTP

    7. 5 µl 10 mM rCTP

    8. 1 µl RNase Inhibitor

    9. 1 µl T7 polymerase

  2.  Vortex, spin briefly

  3.  Add 10 µl template DNA, mix, spin briefly

  4.  Incubate for 1 hour at 40°C

C. Destroy DNA, purify RNA

  1.  Add 1 µl RQ1 RNase-free DNase

  2.  Incubate for 15 minutes at 40°C

  3.  Add 106 µl ddH2O

  4.  Sequentially add the following:

    1. 200 µl Phenol (water-saturated)

    2. 40 µl Chloroform

  5.  Vortex, spin for 5 minutes at 14,000 rpm

  6.  Pass the upper aqueous phase through G25 column

  7.  Precipitate with:

    1. 1 µl linear acrylamide

    2. 30 µl 3M Sodium Acetate pH5.2

    3. 600 µl 100% Ethanol

  8.  Mix, precipitate at -80°C for 15 minutes.

  9.  Spin 15 minutes at 14,000 rpm

  10.  Wash pellet with 70% Ethanol

  11.  Wash pellet with 100% Ethanol

  12.  Dry pellet for 10 minutes

  13.  Dissolve pellet in 11 µl ddH2O

  14.  Use 1 µl to determine RNA concentration

Svetlana Mikheeva, 13 August 1999

Department of Microbiology
School of Medicine
University of Washington
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