PEG-Plasmid-Max-Prep

1. Inoculate 500 mL 2xYT or LB/Amp or Kan from 1-5 mL overnight

2. Grow at 37 ºC overnight.

3. Spin cells at 6000 rpm for 10 min at 4ºC using GS3 rotor.

4. Resuspend cells in 40 mL P1.

5. Add 40 mL P2. Stir very gently using a long 5 ml plastic pippette, until homogenous and clear. Put on ice for 10 min.

6. Add 56 mm P3 with Ca, mix, put on ice for 10 min.

7. Spin 6000 rpm at 4ºC for 10 min.

8. Filter supernatant using autoclaved cheesecloth or coffee filter paper.

9. Collect filtrate to a clean centrifuge tube.

10. Add 0.6 volume isopropanol or 2 volume of ethanol. Invert to mix. Let it for precipitate at room-temperature for 10 min.

11. Spin at 6000 rpm at 4ºC or 25 ºC for 10 min.

12. Wash pellet with 70% ethanol, votex, spin for 5 min, decant ethanol.

9. Add CTAB solution to filtrate at final concentration 0.2%, spin at 6000 rpm for 10 min.

13. Resuspend pellet in 5 ml autoclaved 0.75 M NaCl, wash tube with 2.5, 2.5 mL of 0.75M NaCl (wash 2 times), combine liquid and transfer to a clean tube.
optional: add 20 ÎŒg RNase A and incubate 37ºC 20 min to digest residual RNA.

14. Spin 12000 rpm, collect supernatant---> plasmid DNA solution

15. Add 10% Triton X-114 to final concentration of 1% TritonX-114 to the plasmid DNA solution.

16. Incubate on ice for 10 min, aliquot to 1.5 ml tubes

17. Incubate in a 42°C heating block waterbath for 10 min.

18. Spin at RT at max speed (Eppendorf) 6000 rpm for 5 min.

19. Collect (and combine) supernatant, add 0.43 mL PEG/NaCl/MgCl₂ solution per 1 mL supernatant. (Add MgCl₂ to final concentration of 10 mM).

or add 110 µl of Ca/PEG to 1 ml of supernatant.

Mix and let incubate at RT for 10 min.

20. Spin at 12000 rpm  for 10 min (SA-600 rotor).

21. Wash with 70% ethanol.

22. Spin at 12000 for 1 min.

23. Remove ethanol and air-dry in a biosafety hood.

24. Add 1 ml water to dissolve DNA.

25. Measure DNA concentration and OD260/280 using NanoDrop and run 5 ul on agarose gel to check for the presence of RNA.

TE (Tris/EDTA) [500 mL]
25 mM Tris•HCl pH 8.0 [6.25 mL 2M]
10 mM EDTA [10 mL 0.5M]
Autoclave

P2 (~5.5) [500 mL]
147g KOAc (3M)
55 mL glacial HOAc

N3 with Ca

4.2 M Gu-HCl,
0.9M KAc,
0.4 M CaCl₂,

2.0M HAc (pH 4.8)

PEG/NaCl/MgCl₂ solution
30% w/v PEG 8000
1.6M NaCl
25 mM MgCl₂.

Ca/PEG
0.4 M calcium chloride
40% PEG 8000900 ul of plasmid DNA in EB, + 0.3 ml 4 M NaCl, + 0.3 ml 50% PEG, spin 10 min.