碱裂解法制备大肠杆菌质粒（Alkaline Lysis Plasmid Preparation from E.

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This protocol yields plasmid DNA that is suitable for restriction digests and cloning purposes. This preparation method works well on all E. coli strains and also exponentially growing Rhizobium meliloti cultures.
1. Grow a 2-3 ml culture in rich media with appropriate antibiotic selection. (10 ml culture of R. meliloti)
2. Pellet 1.5 ml in a microfuge tube for 30 sec-2 min. (10 ml for R. meliloti)
3. Pour off supernatant and vortex pellet until a homogenous Cell paste is obtained.
4. Add 0.2 ml freshly made Lysozyme solution, mix gently and incubate on ice for 5 min. Lysozyme solution: 1 ml GEundefined + 5 mg lysozyme
5. Add 0.4 ml of freshly made NaOH - SDS solution. Mix gently by rocking tube. The solution should turn translucent as Cell s lyse. Incubate on ice 5 min.
NaOH - SDS solution (per ml): 0.7 ml water
0.2 ml 1 M NaOH
0.1 ml 10% SDS
6. Add 0.3 ml Potassium Acetate Stocundefined_and_vortex_gently._A_precipitate_should_form.~Kbr_~H~M~2~1~0~0Can_be_frozen_at_this_point_to_increase_yield_but_not_required.~Kbr_~H~M~2~1~0~07._Centrifuge_for_10_min_on_maximum.~Kbr_~H~M~2~1~0~08._Carefully_remove_0.75_ml_of_the_supernatant_and_transfer_to_a_clean_and_labeled_microfuge_tube._Be_careful_not_to_take_any_material_at_the_interface._Add_0.45_ml_~A0.6_volume~B_of_isopropanol_and_mix_well_using_the_vortexer.~Kbr_~H~M~2~1~0~09._Centrifuge_for_10_min_on_maximum.~Kbr_~H~M~2~1~0~010._pour_off_supernatant_and_wash_briefly_with_0.4_ml_cold_70~7_ethanol_~Arock_tube_5_times~B._Pour_off_ethanol_wash_and_spin_30_sec_in_microfuge._Remove_remaining_liquid_with_a_micropipet_and_dry_pellet_in_a_vacuum.~Kbr_~H~M~2~1~0~011._Dissolve_pellet_in_20_μl TE buffer. Use one to three microliters for restriction digests.
Store plasmid prep at 4° for days-weeks or -20° for months-years.
GEundefined: 50 mM glucose, 10 mM EDTA , 25 mM Tris-HCl pH 8.0
Potassium Acetate Stocundefined~I~Kbr_~H~M~2~1~0~060_ml_5_M_potassium_acetate~Kbr_~H~M~2~1~0~028.5_ml_glacial_acetic_acid~Kbr_~H~M~2~1~0~011.5_ml_water~Kbr_~H~M~2~1~0~0~ApH_4.8~B~Kbr_~H~M~2~1~0~0store_at_room_temperature~K~Hp~M~2~1~K~Hdiv~M~2~1