Team:Glendale CC AZ/Protocols/AlkalineLysis

Glendale Community College Arizona

Protocols

Alkaline Lysis Plasmid Miniprep Protocol

Purpose: To isolate the plasmid DNA from transformed cells.

Reagents:

Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)

  -9.0mL dH2O
  -0.25mL 1M TrisHCl pH 8
  -0.25mL 0.5 M Na2EDTA
  -0.50mL 20% glucose

SDS/NaOH (prepare fresh)

  -880µl dH2O
  -100µl 10% SDS
  -20µl 10M NaOh

Acetate solution

  -60mL 5M potassium acetate
  -11.5mL glacial acetic acid
  -28.5mL dH2O

Materials:

       -1 colony of transformed cells
       -5mL LB/chloramphenicol media
       -Microcentrifuge
       -Microcentrifuge tubes
       -Micropipette
       -Disposable tips
       -200µl lysis solution
       -400µl SDS/NaOH (made fresh)
       -300µl acetate solution
       -1000µl isopropanol
       -400µl 70% ethanol
       -Kimwipes
       -100µl TE buffer

Procedure:

1. Inoculate 5ml of LB/antibiotic (chloramphenicol, or other) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.

2. Optional: Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.

3. Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.

4. Add 400µl of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.

5. Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.

6. Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.

7. Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.

8. Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.

9. Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.

10. Discard supernatant, removing residual liquid with pipet tip. Add 200-400ul 70% ethanol, agitate pellet gently and centrifuge at 10,000 rpm for 10 min to precipitate pellet.

11. Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.

12. Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55µl. Optional: Let pellet hydrate overnight.