Isolation and Purification of Plasmid DNA from E . coli
Inoculate 1000 mL of L broth containing the appropriate antibiotic with 10 mL of an overnight culture of the E . coli strain containing the plasmid to be purified .
Incubate at 37°C overnight .
If amplification of the plasmid DNA is necessary ( because of low copy number of the plasmid under normal conditions ) , after 4 - 8 hours of growth , add chloramphenicol ( 34 mg / mL ) to a final concentration of 170 ug / mL or spectinomycin ( 10 mg / mL ) to a final concentration of 50 ug / mL ( if the bacteria carries chloramphenicol resistance ) .
Incubate at 37°C overnight .
Centrifuge the bacterial cells in the Sorvall GSA rotor at 5,000 rpm , 5 min , 4°C .
Use 3 - 250 mL GSA bottles per liter of bacteria .
Discard the supernatants .
Resuspend the cells with 60 mL ( total , 20 mL / bottle ) of lysozyme solution .
Incubate on ice for 30 min .
Add 120 mL ( total , 40 mL / bottle ) of the alkaline SDS solution .
Gently mix ( by inversion ) until the solution clears .
Incubate on ice for 5 min .
Add 90 mL ( total , 30 mL / bottle ) of the high salt solution .
Mix well and incubate on ice for 30 - 60 min .
Centrifuge the bottles in the Sorvall GSA rotor at 10,000 rpm , 10 min , 4°C .
Decant the supernatants to clean bottles .
Precipitate the DNA with 2X volumes ( approximately 170 - 180 mL ) of 100 % EtOH .
Hold at - 80°C for 60 min .
Centrifuge the bottles in the Sorvall GSA rotor at 10,000 rpm , 10 min , 4°C .
Discard the supernatants .
Resuspend the pellets with 10 mL each of 50 mM Tris - HCl , pH 8.0 , 100 mM NaOAc .
Transfer the material to 30 mL corex tubes .
Add an equal volume of buffer - saturated phenol to each tube .
Mix well and centrifuge in the Sorvall SS34 rotor at 10,000 rpm , 10 min , room temperature .
Remove the upper aqueous layers to clean 30 mL corex tubes .
Precipitate the DNAs with 2X volumes of 100 % EtOH .
Hold at - 80°C for 45 min .
Centrifuge the tubes in the Sorvall HB - 4 rotor at 10,000 rpm , 10 min , 4°C .
Discard the supernatants .
Dry the pellets briefly ( 10 - 15 min ) in the vacuum desiccator to remove the EtOH .
Resuspend each plasmid DNA with a total of 10 mL of 1X TE buffer .
Weigh out exactly 6.8 gm of CsCl into each of 2 - 30 mL corex tubes for each plasmid DNA .
Divide each plasmid DNA solution between the 2 tubes of CsCl , measuring the volume .
Add 1X TE buffer to the tubes of CsCl to a final volume of 6.4 mL added to each tube .
Mix well , until all the CsCl is in solution .
Add 512 µL of ethidium bromide ( 10 mg / mL ) to each tube .
Mix well .
Centrifuge the tubes in the Sorvall SS34 rotor at 10,000 rpm , 15 min , 4°C .
Transfer the supernatants to Beckman polyallomar Ti50 rotor quick - seal tubes using a syringe and needle ( 18g ) .
Fill the tubes to capacity with a CsCl / ethidium bromide solution ( 6.8 gm CsCl , 6.4 mL 1X TE buffer , 512 µL 10 mg / ml ethidium bromide ) .
Seal the tubes with the Beckman tube sealer .
Centrifuge the gradients in the Beckamn Ti50 rotor at 35,000 rpm , 72 hours , 25°C .
Visualize the DNA using a UV light source .
Remove the plasmid DNA from the gradients by puncturing the side of the tube with an 18g needle and syringe .
Place a small amount of Vaseline on the side of the tube and puncture through the Vaseline .
Smear the Vaseline over the puncture hole when the syringe is removed to seal the hole .
Circular plasmid DNA is the lower band in the gradient .
Chromosomal DNA and nicked circular DNA is in the upper band in the gradient .
Place the DNA solution into 30 mL corex tubes .
Extract the ethidium bromide from the DNA solution by adding an equal volume of CsCl / TE buffer saturated isopropanol .
Mix gently and centrifuge in the Sorvall SS34 rotor at 3,000 rpm , 1 min , 4°C .
Pipet off the upper isopropanol layer .
Repeat the isopropanol extraction 1X .
Add 1.0 mL of 3 M NaOAc to each tube of DNA and adjust the volume in each tube to 10.0 mL with 1X TE buffer .
Precipitate the DNAs with 2X volumes of 100 % EtOH .
Hold at - 20°C overnight .
Centrifuge the tubes in the Sorvall HB - 4 rotor at 10,000 rpm , 10 min , 4°C .
Discard the supernatants .
Dry the pellets briefly ( 10 - 15 min ) in the vacuum desiccator to remove the EtOH .
Resuspend the plasmid DNAs with 1X TE buffer .
Determine the DNA concentration .
Store the DNAs at 4°C .
