Posted on June 2nd, 2016 by UNM CC
M. Fero — 12/29/00
- Grow 250 500 mL of bacteria overnight in LB with 50 µg/mL of ampicillin.
- Transfer culture to Nalgene bottles. Spin in SGA rotor at 6000 rpm, at 4°C for 10 min.
- Discard supernatant. Keep everything on ice. Resuspend bacterial pellets by adding 15 mL of water and split into 2 plastic Sorval tubes (max capacity = 40 mL/tube)
- Add 17.5 mL of fresh Solution II to each tube. Mix by inversion. Keep on ice for 10 min.
- Add 13 mL of Solution III. Mix by sharp inversion. Ice for 20 min.
- Spin for 20 min. at 8,000 rpm (12,000g) in a JA-10 rotor.
- Transfer supernatant to a 50 mL conical tubes by filtering through Kimwipes, use glass pipettes and avoid white cheesy gunk.
- Split evenly into 4 glass Sorval tubes (max 20 mL/tube) Add an equal volume of isopropanol to each tube. Mix.
- Spin 15 min. at 8,000 rpm (12,000g) in a JA-10 rotor.
- Discard supernatant. Air dry pellet.
- Resuspend pellets in a total of 4 mL of T.E. with 100 µg/mL of boiled RNAseA and transfer to 15 mL conical polypropylene tubes. Incubate at 25°C for 30 min. Add 0.5 mL of 5 M NaCl and 100 µL of 10% SDS, mix.
- Extract 2x with an equal volume of phenol. Extract 1x with CHCl3 (with 1/25 v/v isoamyl alcohol. Repeat the extractions if there is still gunk left in the aqueous phase.
- Transfer aqueous phase to 15 mL Corex tubes, add 9.4 mL of cold ethanol, mix. (It's OK to store the samples in the freezer at this point)
- Centrifuge at 10,000 rpm, 10 min, 4°C.
- Resuspend pellet in 500 µL of TE. Add 120 µL 5M NaCl, mix. Add 400 µL of 20% PEG 8000. Incubate on ice for 20 min. Pellet at 14,000 rpm, 10 min.
- Discard supernatant. Pulse spin and discard remainder of supernatant.
- Resuspend in 200 µL of TE. Add 200 µL 5M NH4OAC. Ice 20 min. Spin at 14,000 rpm. Save supernatant.
- Add 1 mL of cold EtOH. Incubate at 70°C for 5 10 min. or longer at 20°C.
- Rinse 2x with 70% EtOH. Air dry pellet.
- Resuspend in 500 µL of TE. Check A260 and A280 and inspect on agarose gel.
- Store glycerol stocks of the bacteria and make plasmid maps if they are not already done.
- Solution II: 0.2M NaOH, 1% SDS. (Make fresh. For 20 mL add 0.4 mL 5M NaOH, plus 2 mL 10%SDS)
- Solution III: 3M KOAc, pH4.8. (60 mL KOAc, 11.5 mL glacial HOAc, 28.5 mL water).
- Isopropanol, Ethanol, 70% ethanol
- RNAseA: 10 mg/mL in water (boil for 20 min. Spin to remove debris. Freeze in aliquots)
- Phenol, equilibrated with TE, pH8
- Chloroform (+ Isoamyl alcohol 1/25 v/v).
- 20% PEG 8000