Miniprep - GET Buffer protocol

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Solutions/reagents:

• overnight culture
• ice-cold GET buffer
  
  (50 mM glucose (MW 180), 10mM EDTA, 25 mM Tris-HCl pH 8)
• 0.2M NaOH stored at room temperature
• ice-cold potassium acetate solution
  
  (3 M potassium acetate, 1.8 M acetic acid, no pH adjustment)
• 95% / 100% ethanol
• 70% EtOH
• TE buffer
• distilled water
• PCA solution
  
  ((optional)50 parts phenol, 49 parts chloroform, and 1 part amyl-alcohol)
• SDS
• lysozyme
  (optional)
  

Equipment:

• Centrifuge
• Sterile 2-ml microcentrifuge tubes

Steps:

1. Measure out 2 ml of overnight culture into sterile 2-ml microcentrifuge tube (1).
   
2. Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
   
3. Measure out 100 µl of ice-cold GET buffer into sterile 2-ml microcentrifuge tube (1).
   Resuspend pellet by vortexing/by shaking vigorously.
   

(Optional)
Add 10 mg of lysozyme.
Store at room temperature for 30 mins.
This step is essential for lysing gram-positive cells.


4. Measure out 200 µl of 0.2M NaOH into sterile 2-ml microcentrifuge tube (2).
   Add 2 mg of SDS.
   Vortex the mixture for a few secs.
   
5. Measure out alkaline SDS solution into sterile 2-ml microcentrifuge tube (1).
   Close the tube tightly and gently mix the contents by inverting the tube.
   DO NOT VORTEX! The solution should become clear.
   
6. Add 150 µl of ice-cold potassium acetate solution.
   Close the tube tightly and gently mix the contents by inverting the tube.
   DO NOT VORTEX! A precipitate should form.
   
7. Store the tube on ice for 3 - 5 mins.
   
8. Centrifuge at maximum speed for 10 mins at room temperature and aspirate out 400 µl of top layer.
   Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (3).
   Discard bottom layer.
   DO NOT PICK UP ANY PRECIPITATE!!!
   

(Optional)
Measure out 400 µl of PCA solution into sterile 2-ml microcentrifuge tube (3).
Close the tube tightly and gently mix the contents by inverting the tube.
Centrifuge at maximum speed for 3 mins at room temperature and aspirate out the top layer.
Transfer top aqueous layer into sterile 2-ml microcentrifuge tube (4).
Discard bottom layer.
This helps remove any residual proteins.


9. Measure out 900 µl of 95% / 100% ethanol into sterile 2-ml microcentrifuge tube (4).
   This is to precipitate the plasmid DNA.
   
10. Store at -80°C for 30 mins.
    Use the -80°C freezer.
    
11. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
    
12. Add 1 ml of 70% EtOH.
    Store at room temperature for 3 mins.
    
13. Centrifuge at maximum speed for 3 mins at room temperature, gently aspirate out the supernatant and discard it.
    Make sure the pellet is toward the outside.
    
14. Dry the pellet in air for 10 - 15 mins.
    
15. Make sure the pellet is completely dry before this step.
    

    Option 1: Add 20 µl of TE buffer.
    (or)
    Option 2: Add 20 µl of distilled water.
    

    Resuspend pellet by vortexing/by shaking vigorously.
    The DNA will contain RNA contamination, which can be removed by resuspending in TE with RNAse.
    

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 50 mins