Plasmid minipreps. So basic, so simple, so beautiful!

This is how we purify plasmid DNA!  No kits please!

1)      Inoculate bacterial culture aseptically in 3 ml LB (or similar media) with the appropriate antibiotics.  Incubate over night with agitation (250-300 rpm) @ 37 ^oC.

           Inoculation means picking a colony with an inoculation loop.  Or if you don`t have one, take sterile forceps and pick a sterile pipette tip or tooth stick or anything else that is sterile and have a fine tip.  Tap onto the colony and drop it into the tube with LB.  Replica plates can also be produced at this step, just scratch the surface of a fresh plate with loop/pipette tip/tooth stick and then drop it into the tube.  And once you are already at making replica plates, why not pool the minipreps? When working with difficult ligations, we pool three colonies per tube. Just make sure you replicate each of them on the replica plate with proper labeling!

2)      Transfer 2 ml of the over night culture into 2 ml Eppendorf tube.

Also works with 1.5 ml culture in 1.5 ml tubes.  Transfer means pouring, you don`t have to keep this sterile.

3)      Pellet the cells by centrifugation (~7-8000 g, 1 min).

4)      Remove supernatant and re-suspend cells in 300 μl P1 by vortexing.

5)      Add 300 μl P2.  Turn the tubes upside down and back 8 times.  (Do not vortex or shake too hard!  You can add solution first to each tube, close each tube and then make the mixing).

6)      Let the tubes stand on the bench for 5 mins.

7)      Add 300 μl P3, close the cap, shake the tube and place it on ice. (Shake the tube vigorously immediately after adding solution, but do not vortex!)

8)      Let the tubes stand on ice for 10 mins.  (5 mins is enough when you are on a rush.  You can also extend this incubation for up to an hour.)

9)      Centrifuge at 12000 g for 10 mins.

10)  Transfer supernatant into a new 1.5 ml Eppendorf tube.

11)  Add 200 μl phenol-chlorophorm, mix by vortexing.

12)  Centrifuge at 12000 g for 10 mins.

13)  Transfer supernatant into a new 1.5 ml Eppendorf tube.

14)  Add 500 μl i-propanol.  Mix and let the tube stand on the bench for 5 mins.

15)  Centrifuge at 12000 g for 10 mins.

16)  Remove supernatant and add 500 μl 70% Et-OH.

17)  Centrifuge at 12000 g for 10 mins.

18)  Remove supernatant and dry the pellet.

19)  Add 50 μl ultra pure water with 10 μg / ml RNase.

Solutions

P1

Dissolve 6.06 g Tris base, 3.72 g Na₂EDTA.2H₂O in 800 ml distilled water. Adjust the pH to 8.0 with HCl. Adjust the volume to 1 liter with distilled water.

Optionally 100 mg RNase A per liter can be added. Store at 4^oC.

P2

Dissolve 8 g NaOH and 10 g SDS (1 m/w %) in 1 liter final volume of water. Store at room temperature.

P3

Dissolve 294.5 g potassium acetate in 500 ml distilled water. Adjust pH to 5.5 with glacial acetic acid (110-130 ml). Adjust volume to 1 liter with distilled water. Store at 4^oC.