Alkaline Lysis Plasmid Miniprep

Protocol

1. Inoculate 2 ml 2X YT media (supplemented with the appropriate antibiotic) with a medium sized bacterial colony. Incubate at 37°C overnight.
2. Transfer 1.5 ml of the bacterial suspension to a 1.5 Epindorf tube. Spin for 20 seconds in a microfuge. Remove the supernatant using a P200.
3. Resuspend the pellet in 110 µl of Solution I. Complete dispersion of the pellet is critical.

   Solution I
   [Stock]	Reagent	Amount	[Final]
   2 M	Glucose	2.5 ml	50 mM
   1 M	Tris-HCl (pH = 8)	2.5 ml	25 mM
   0.5 M	EDTA (pH = 8)	2 ml	10 mM
   --	H2O	93 ml	--
   Autoclave

4. Let sit 5 minutes at room temperature.
5. Add 200 µl of Solution II. Mix gently and incubate on ice for 10 minutes.

   Solution II
   [Stock]	Reagent	Amount	[Final]
   10 N	NaOH	0.2 ml	200 mN
   20 %	SDS	0.5 ml	1 %
   --	H2O	9.3 ml	--

6. Add 150µl ice-cold Solution III. Vortex at the highest setting for 2 seconds to mix. Place on ice for at least 5 minutes.

   Solution III
   Reagent	Amount
   KOAc	29.44 g
   Glacial Acetic Acid	11.5 ml
   H2O	to 100 ml
   Dissolve the KOAc in ~50 ml H2O, add the acetic acid then add H2O to reach 100 ml. Store at -20°.

7. Spin for 5 minutes in a microcentrifuge to pellet the cell debris and chromosomal DNA. Repeat if necessary.
8. Transfer the supernatant to a new 1.5 ml Epindorf tube. Add 450 µl phenol. Vortex 20 seconds to 1 minute. Spin in the microcentrifuge for 10 minutes.
9. Transfer the aqueous (top) phase to a new 1.5 ml Epindorf tube. Extract with an equal volume of chloroform:isoamyl alcohol (24:1). Spin for 5 minutes in the microcentrifuge. Transfer the aqueous (top) phase to a new 1.5 ml Epindorf tube.
10. Add two volumes 95% ethanol. Incubate for at least 20 minutes at -20°C.
11. Spin in the microcentrifuge for 10 minutes. Decant the supernatant and wash the pellet once with 70% ethanol.
12. Dry the pellet in the SpeedVac for 10 minutes.
13. Resuspend the pellet in 30 µl TE.
14. In digestions use 1 µl of the plasmid prep per reaction.