In vitro Transcription


    20 µl 1X Reaction - Use this when making RNA pools containing 96 clones

    2 µl linearized DNA

    4 µl H2O

    10 µl 2X NTP/CAP

    2 µl 10X Reaction Buffer

    2 µl Enzyme Mix

    20 µl           2 hours @ 37°C


    or


    10 µl 1X Reaction    - Use this when making RNA for split pools

    1 µl linearized DNA

    2 µl H2O

    5 µl 2X NTP/CAP

    1 µl 10X Reaction Buffer

    1 µl Enzyme Mix

    10 µl           2 hours @ 37°

     

     

  1. Add 1 µl or 0.5 µl DNAse I and mix well, incubate at 37°C for 15 min.

  2. Stop reaction and precipitate the RNA by adding 25 µl or 12.5 µl LiCl, place in -20°C freezer for at least 30 min.

  3. Spin down 15 min., wash with 70% EtOH

  4. Re-suspend RNA in 20 µl or 10 µl DEPC H2O

  5. Add 2 µl or1 µl RNase free 3 M NaOAc ph 5.2

  6. Add 50 µl or25 µl of 100% EtOH, put in -20°C for 30 min.

  7. Spin 15 min. at max in centrifuge

  8. Pour off supernatant, wash with 1 µl 70% EtOH

  9. Vortex, spin 5 min. @ max speed, pour off supernatant

  10. Re-suspend in 21 µlor 11 µl DEPC H2O

  11. Add 1 µl to RNA loading dye, heat at 65°C for 5 min., check on gel.

    12.