Large scale prep:
1) Spin down cells and resuspend in 5ml (for a 50-200ml culture) of 25mM Tris, 10mM EDTA pH 8.0.
2) Add 10ml of freshly prepared 0.2M NaOH, 1% SDS and mix thoroughly. Leave for 2-3min. MAXIMUM.
3) Add 7.5ml KoAc solution. Vortex.
4) Spin 10 min. at 7K in Sorval.
5) Pour supernatant through a piece of cheese cloth and add 11ml isopropanol. Spin 10 min. 7K in Sorval (the large amount of RNA acts as a carrier).
6) Resuspend pellet in 3ml of TE. Add to tube and make up to 4.3ml with TE. Add 100µl of 10mg/ml ethidium bromide and 4.4g of CsCl.
7) Band for 6hr (minimum) to o/n in VTi65 at 50-55K 20°C.
8) +/- reband by pulling band and adding to fresh tube and topping up with CsCl density 1.6 (is 1g CsCl per ml TE, no extra E'Br required). Spin as above.
9) Remove ethidium bromide by extracting with saturated butanol 3-4 times. Isolate plasmid DNA by adding an equal volume of TE and 5 volumes of room temperature ethanol. Spin down plasmid immediately and resuspend in 400µl TE. Reprecipitate in an ependorf tube by adding 1ml ethanol and 35µl 5M NaCl.
Mini preps ("1 tube method")
mini-prep;
1) Grow overnight of 1.5ml.
2) Pour into eppendorf tube and spin down cells at 7-8K for 2min (low speed makes them easier to resuspend).
3)Aspirate supernatant and resuspend cells in 50µl 25mM Tris pH 8.0, 10mM EDTA. You can leave the lids off for all the following steps till you add the phenol.
4) Add 100µl of (freshly prepared) 1% SDS, 0.2M NaOH. Add it forcefully and you don't need to vortex. It is not necessary to pause between steps 4, 5, 6.
5) Add 75µl KOAc solution and vortex.
6) Add 100µl of phenol/CHCl3. Close lids and vortex.
7) Spin in ependorf 13K for 2min.
8) Add supernatant to 500µl ethanol and spin 13K for 5min.
9) Aspirate supernatant, removing all the ethanol. Resuspend in 50µl TE (you don't need a 70% rinse unless you are going to cut with a very salt sensitive enzyme).
10) Dispose of phenol into appropriate waste before discarding tubes.
I normally digest 2-5µl of this prep for an analytical gel. This DNA contains very little
chromosomal and is fine for cloning.
KOAc solution is:
60ml 5M potassium acetate
11.5ml glacial acetic acid
28.5ml of DDW.
Not necessary to pH it (it should be around pH 6).
No phenol method according to Gilles Morelle in Focus (11.1). At step 5 of mini-prep.
Add 75µl 7.5M ammonium acetate instead of KOAc (pH 7.8 without adjustment)
and then leave the tube on ice for 10 min.. The RNA and chromosomal will
precipitate. Spin 3min 10K and add 0.6vol. (100-125µl) isopropanol, leave on ice 10
min. and spin 15K 10 min., 70% rinse and resuspend pellet in 100µl TE.