Erman's Lab:DNA Miniprep with Alkaline Lysis protocol

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Solutions/reagents:

• LB (+ antibiotics)
• Alkaline Lysis SLN1
  
  (20mM Tris(pH 8), 50mM Glucose, 10mM EDTA)
• freshly prepared AL2
  
  (0.2N NaOH, 1% SDS)
• AL3
  
  (3M KAc, glacial acetic acid)
• isopropanol stored at room temperature
• 70% EtOH
• TE
• water
• single colony

Equipment:

• Incubator
• Centrifuge
• Eppendorf tubes

Steps:

1. Inoculate 2 ml LB (+ antibiotics) with single colony and incubate with shaking for 12 hrs(overnight) at 37°C.
   
2. Measure out 2 ml of culture into Eppendorf tube (1).
   
3. Centrifuge at maximum speed for 1.5 mins at 4°C, gently aspirate out the supernatant and discard it.
   
4. Leave pellet as dry as possible.
   
5. Add 100 µl of Alkaline Lysis SLN1.
   Resuspend pellet by vortexing/by shaking vigorously.
   
6. Add 200 µl of freshly prepared AL2.
   Close the tube tightly and invert the tube 5 - 6 times.
   Do not vortex!
   
7. Store the tube on ice.
   
8. Add 300 µl of AL3.
   Close the tube tightly and gently mix the contents by inverting the tube.
   
9. Incubate on ice for 5 mins.
   
10. Centrifuge at maximum speed for 5 mins at 4°C and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (2).
    Discard bottom layer.
    
11. Measure out 900 µl of isopropanol into Eppendorf tube (2).
    Vortex the mixture for a few secs.
    
12. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
    
13. Add 1 ml of 70% EtOH.
    Vortex the mixture for a few secs.
    
14. Centrifuge at maximum speed for 5 mins at room temperature, gently aspirate out the supernatant and discard it.
    
15. Dry the pellet in air.
    

16. Option 1: Add 50 µl of TE.
    (or)
    Option 2: Add 50 µl of water.
    

    Dissolve the pellet in the solution.
    

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 26 mins