BACDNA分离方法IsolationofBACDNAfromLargescaleCultures

Isolation of BAC DNA from Large-scale Cultures

• Alkaline lysis solution I, ice coldPrepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4°C.
   
  

• 50 mM glucose

• 25 mM Tris-Cl (pH 8.0)

• 10 mM EDTA (pH 8.0) 
   
  

• Alkaline lysis solution II, freshly prepared (room temperature)Solution II should be freshly prepared and used at room temperature.
   
  

• 0.2 N NaOH (freshly diluted from a 10 N stock)

• 1% (w/v) SDS 
   
  

• Alkaline lysis solution III, ice cold

• 5 M potassium acetate, 60.0 ml

• glacial acetic acid, 11.5 ml

• H₂O, 28.5 ml

• Ethanol

• Ethanol (70%)

• Isopropanol

• Phenol:chloroform (1:1, v/v)

• Sodium acetate (3 M, pH 5.2)

• STE, ice cold

• 10 mM Tris-Cl (pH 8.0)

• 0.1 M NaCl

• 1 mM EDTA (pH 8.0)

• TE (pH 8.0)

• 100 mM Tris-Cl (desired pH)

• 10 mM EDTA (pH 8.0)

• E. coli strain carrying a recombinant BAC

• LB containing 12.5 µg/ml chloramphenicol

• Lysozyme

• Pancreative RNase (RNase A), free of DNase

• Restriction enzymes and 10x buffers

• DNA markers for pulsed-field gel electrophoresis

• Pulsed-field gels (or 0.5% agarose gels)

• Sorvall SLC-1500rotor and Oak Ridge tubes (4°C)

• Bacterial shaker (37°C)

• Chromatography resin (optional; see Step 13)

• Microfuge (4°C)

• Sterile 17 x 100-mm tubes

1. Inoculate 500 ml of LB medium containing 12.5 µg/ml of chloramphenicol with 50 µl of a saturated overnight culture of BAC-transformed cells. Incubate the 500-ml culture for 12-16 hours at 37°C with vigorous agitation (300 cycles/minute) until the cells reach saturation. 
   

2. Harvest the cells from the culture by centrifugation at 2500g (3900 rpm in a Sorvall SLC-1500 rotor) for 15 minutes at 4°C. Pour off the supernatant and invert the open centrifuge bottle to allow the last drops of the supernatant to drain away. 
   

3. Resuspend the bacterial pellet in 100 ml of ice-cold STE. Collect the bacterial cells by centrifugation as described in Step 2. 
   

4. Resuspend the bacterial pellet in 24 ml of alkaline lysis solution I containing DNase-free RNase (100 µg/ml). Add lysozyme to a final concentration of 1 mg/ml. 
    
   Make sure that the cells are completely resuspended and that the suspension is free of clumps.  
   

5. Add 24 ml of freshly prepared alkaline lysis solution II. Close the top of the centrifuge bottle and mix the contents thoroughly by gently inverting the bottle several times. Incubate the bottle for 5 minutes on ice.
    
   

6. Add 24 ml of ice-cold alkaline lysis solution III. Close the top of the centrifuge bottle and mix the contents gently but thoroughly by swirling the bottle until there are no longer two distinguishable liquid phases. Place the bottle on ice for 5 minutes.
    

7. Centrifuge the bacterial lysate at 15,000g (9600 rpm in a Sorvall SLC-1500 rotor) for 10 minutes at 4°C. At the end of the centrifugation step, decant the clear supernatant into a polypropylene centrifuge bottle. Discard the pellet remaining in the centrifuge bottle. 
   

8. Add an equal volume of phenol:chloroform. Mix the aqueous and organic phases by gently inverting the tube several times. Separate the phases by centrifugation at 3000g (4300 rpm in a Sorvall SLC-1500 rotor) for 15 minutes at room temperature. 
   

9. Use a wide-bore pipette to transfer the aqueous layer to a fresh centrifuge bottle and add an equal volume of isopropanol. Invert the bottle several times to mix well. 
   

10. Mark the tube on one side and place it in a centrifuge rotor with the marked side facing away from the center of the rotor. Marking in this way will aid in the subsequent identification of the nucleic acid pellet. Recover the precipitated nucleic acids by centrifugation at 15,000g (9600 rpm in a Sorvall SLC-1500 rotor) for 15 minutes at room temperature.
     
    

11. Decant the supernatant carefully and invert the bottle on a paper towel to allow the last drops of supernatant to drain away. Rinse the pellet and the walls of the bottle with 20 ml of 70% ethanol at room temperature. Drain off the ethanol and place the inverted tube on a pad of paper towels for a few minutes at room temperature to allow the ethanol to evaporate. 
    

12. Gently dissolve the pellet of BAC DNA in 0.2 ml of TE (pH 8.0). Assist in the dissolution of the DNA by tapping the sides of the bottle rather than vortexing. Measure the concentration of DNA by absorption spectroscopy. 
    

13. Digest the BAC DNA with restriction endonucleases. 
    If the DNA preparation is resistant to cleavage, further purification by column chromatography on Qiagen resin usually eliminates the problem. 
    

14. Analyze the digested BAC DNA by pulsed-field gel electrophoresis, using DNA markers of an appropriate size.