Linearizing plasmid pools for in vitro transcription

Digest 20 µl DNA (plasmid pool) with 2 ml Asc I in 100 µl Reaction

1X Reaction

20 µl DNA

10 µl Buffer 4

2 µl Asc I

68 µl H2O

100 µl  2 hours @ 37°C

  1. To each tube add 97 µl H2O, 2 µl PK (Proteinase K), 1 µl 20% SDS, incubate at 42°C for 20 min.

  2. Add 200 µl phenol/chloroform (bottom phase), vortex and spin 5 min. in centrifuge

  3. Place upper layer into new tube, discard phenol phase

  4. add 20 µl RNase free 3 M NaOAc ph 5.2

  5. Add 2.5-3 volumes of 100% EtOH, put in -20°C for 30 min.

  6. Spin 15 min. at max in centrifuge

  7. Pour off supernatant, wash with 1 mL 70% EtOH

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

  9. Re-suspend in 21 µl DEPC H2O

  10. Check 1 µl on gel