agrobacterium plasmid miniprep (from hiro)
- 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.
- 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.
- discard the supernatant and resuspend the cells in 0.1 ml of sol I.
- add 50 µl of sol I containing 15 mg/ml lysozyme. vortex gently for a few seconds.
- incubate for 10 min at rt.
- add 0.3 ml of freshly prepared sol II and shake to mix.
- incubate for 10 min at rt.
- add 45 µl phenol equilibrated with two volumes of sol II. Vortex gently for a few seconds. It should get very viscous.
- add 225 µl of 3M sodium acetate, pH 4.8. shake the tube briefly.
- incubate at -20ºC for 15 min.
- centrifuge for 3 min at 14k rpm. quickly pour the supernatant into a new 1.5 µl tube.
- fill the tube (~ 700 µl) with 2- propanol (= isopropanol). mix by inverting the tube several times. store at -20ºC for 10 min.
- centrifuge for 5 min at 14 k rpm. discard the supernatant.
- rinse with 75% etoh. dry briefly (1-2 min) under vacuum.
- resuspend the pellet in 150 µl h2o. add 20 µl of 1M mgcl2 and incubate on ice for more than 30 min.
- 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).
- add 400 µl of cold etoh. centrifuge for 5 min at 14k rpm.
- discard the supernatant. rinse with 75% etoh. dry briefly under vacuum.
- 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
|
