Maxiprep of plasmid DNA from E.coli protocol

 

Solutions/reagents:

LB broth + selective marker
50% sterile glycerol
TEG

(25mM Tris-Cl, 10mM EDTA, 50mM dextrose)
20 mg/ml lysozyme
10% SDS
4M NaOH
autoclaved water
Solution 3

(3M potassium-acetate, 2M acetic acid -- glacial is 17M)
isopropanol
70% ethanol
TE buffer
5M LiCl
1 mg/ml RNaseA
phenol: chloroform: isoamyl alcohol

(25:24:1)
chloroform: isoamyl alcohol

(24:1)
straight ethanol
3M sodium acetate
a single colony of E. coli

Equipment:

• Incubator
• Centrifuge
• Eppendorf tubes
• Oakridge tubes

Steps:

 

1. Inoculate 50 ml LB broth + selective marker with a single colony of E. coli and incubate with shaking for 12 hrs (overnight) at 37°C .
   

    

    

2. Measure out 850 µl of culture into Eppendorf tube (1).
   Add 150 µl of 50% sterile glycerol .
   Store at -80°C .
   Measure out 850 µl of culture into Eppendorf tube (2).
   Add 150 µl of 50% sterile glycerol .
   Store at -80°C .
   

    

    

3. Measure out as much culture as will fit into Oakridge tube (1).
   Centrifuge at a speed of 5800 Xg for 10 mins at 4°C , gently aspirate out the supernatant and discard it.
   Add rest of the culture to pellet.
   Centrifuge at a speed of 5800 Xg for 10 mins at 4°C , gently aspirate out the supernatant and discard it.
   Add 1 ml of TEG .
   Resuspend pellet by vortexing/by shaking vigorously.
   

    

    

4. Add 111 µl of 20 mg/ml lysozyme .
   Incubate on ice for 30 mins .
   

    

    

5. Meanwhile:
   Measure out 250 µl of 10% SDS into Eppendorf tube (3).
   Add 125 µl of 4M NaOH .
   Add 2.125 ml of autoclaved water .
   Vortex the mixture for a few secs.
   

    

    

6. Measure out 2 ml of SDS/NaOH mix into Oakridge tube (1).
   Incubate on ice for 10 mins .
   

    

    

7. Add 1.5 ml of Solution 3 .
   Incubate on ice for 10 mins .
   

    

    

8. Vortex the mixture for a few secs.
   Centrifuge at a speed of 17200 Xg for 15 mins at 4°C and aspirate out the top layer.
   Transfer top aqueous layer into Oakridge tube (2).
   Discard bottom layer.
   

    

    

9. Measure out 2.7 ml of isopropanol into Oakridge tube (2).
   Centrifuge at a speed of 17200 Xg for 10 mins at room temperature , gently aspirate out the supernatant and discard it.
   

    

    

10. Add 1 ml of 70% ethanol .
    Vortex the mixture for a few secs.
    Centrifuge at a speed of 17200 Xg for 10 mins at room temperature , gently aspirate out the supernatant and discard it.
    Dry the pellet in air for 2 - 5 mins .
    Add 500 µl of TE buffer .
    Resuspend pellet by vortexing/by shaking vigorously.
    Add 500 µl of 5M LiCl .
    Incubate on ice for 5 mins .
    Centrifuge at a speed of 17200 Xg for 10 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (4).
    Discard bottom layer.
    

     

     

11. Measure out 1 ml of isopropanol into Eppendorf tube (4).
    Incubate at room temperature for 10 mins .
    

     

     

12. Centrifuge at a speed of 17200 Xg for 10 mins at room temperature , gently aspirate out the supernatant and discard it.
    

     

     

13. Add 100 µl of 70% ethanol .
    Vortex the mixture for a few secs.
    Centrifuge at a speed of 17200 Xg for 10 mins at room temperature , gently aspirate out the supernatant and discard it.
    Add 375 µl of TE buffer .
    Resuspend pellet by vortexing/by shaking vigorously.
    Add 7.5 µl of 1 mg/ml RNaseA .
    Incubate at 37°C for 30 mins .
    

     

     

14. Add 700 µl of phenol: chloroform: isoamyl alcohol .
    Vortex the mixture for a few secs.
    The solution should be thoroughly mixed.
    Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (5).
    Discard bottom layer.
    

     

     

15. Repeat protocol from Step 14.
    Repeat until the interface between the phases is clear after centrifugation.
    

     

     

16. Measure out 700 µl of chloroform: isoamyl alcohol into Eppendorf tube (5).
    Vortex the mixture for a few secs.
    The solution should be thoroughly mixed.
    Centrifuge at maximum speed for 2 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into Eppendorf tube (6).
    Discard bottom layer.
    

     

     

17. Repeat Step 16.
    This removes phenol.
    

     

     

18. Measure out 750 µl of straight ethanol into Eppendorf tube (6).
    Add 125 µl of 3M sodium acetate .
    

    Option 1: Store at -80°C for 30 mins .
    (or)
    Option 2: Store at -20°C for 12 hrs (overnight).

     

     

19. Centrifuge at a speed of 13600 Xg for 15 mins at 4°C , gently aspirate out the supernatant and discard it.
    Add ~100 µl of 70% ethanol .
    Vortex the mixture for a few secs.
    Centrifuge at a speed of 13600 Xg for 5 mins at 4°C , gently aspirate out the supernatant and discard it.
    Add 100 - 200 µl of TE buffer .
    Resuspend pellet by vortexing/by shaking vigorously.
    

     

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 15 hrs, 44 mins