LargeScalePlasmidPreps:PEGmethod

1. Grow 250 ­ 500 mL of bacteria overnight in LB with 50 µg/mL of ampicillin.

2. Transfer culture to Nalgene bottles. Spin in SGA rotor at 6000 rpm, at 4°C for 10 min.

3. 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)

4. Add 17.5 mL of fresh Solution II to each tube. Mix by inversion. Keep on ice for 10 min.

5. Add 13 mL of Solution III. Mix by sharp inversion. Ice for 20 min.

6. Spin for 20 min. at 8,000 rpm (12,000g) in a JA-10 rotor.

7. 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.

8. 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.

9. 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.

10. 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.

11. 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.

12. 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.

13. Resuspend in 500 µL of TE. Check A260 and A280 and inspect on agarose gel.

14. Store glycerol stocks of the bacteria and make plasmid maps if they are not already done.

SOLUTIONS:

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