Quint Lab:agrobacterium plasmid miniprep

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agrobacterium plasmid miniprep (from hiro)

  1. pick single colony and grow cells o/n in 3 ml lb medium containing the appropriate antibiotics (usually rif30, gent15-25 for gv3101 and either kan or cam for the plasmid) with vigorous shaking (225 rpm) at 28ºC.
  2. use 900 µl + 100 µl dmso for stock culture and transfer the rest to a 1.5 ml tube. centrifuge the cells for 1 min at 8k rpm.
  3. discard the supernatant and resuspend the cells in 0.1 ml of sol I.
  4. add 50 µl of sol I containing 15 mg/ml lysozyme. vortex gently for a few seconds.
  5. incubate for 10 min at rt.
  6. add 0.3 ml of freshly prepared sol II and shake to mix.
  7. incubate for 10 min at rt.
  8. add 45 µl phenol equilibrated with two volumes of sol II. Vortex gently for a few seconds. It should get very viscous.
  9. add 225 µl of 3M sodium acetate, pH 4.8. shake the tube briefly.
  10. incubate at -20ºC for 15 min.
  11. centrifuge for 3 min at 14k rpm. quickly pour the supernatant into a new 1.5 µl tube.
  12. fill the tube (~ 700 µl) with 2- propanol (= isopropanol). mix by inverting the tube several times. store at -20ºC for 10 min.
  13. centrifuge for 5 min at 14 k rpm. discard the supernatant.
  14. rinse with 75% etoh. dry briefly (1-2 min) under vacuum.
  15. resuspend the pellet in 150 µl h2o. add 20 µl of 1M mgcl2 and incubate on ice for more than 30 min.
  16. centrifuge for 5 min at 14k rpm. transfer the supernatant to a new tube. add 10 µl of 3M sodium acetate, pH 7 (pH 4.8 is fine).
  17. add 400 µl of cold etoh. centrifuge for 5 min at 14k rpm.
  18. discard the supernatant. rinse with 75% etoh. dry briefly under vacuum.
  19. dissolve in 20 µl TE or H2O.

→ for restriction analysis use 5 µl and add 0.5 µl of rnase (2 mg/ml) into the restriction digest.

sol I:

  • 50 mM glucose
  • 25 mM tris pH8
  • 10 mM edta

store at 4ºC

sol II:

  • 1M naoh 200 µl
  • 10% sds 100 µl
  • dh2o 700 µl

prepare fresh, don’t put on ice because of sds