- Transfer a single drug resistant colony to a 50 ml Erlienmeyer flask containing 10 ml 2X YT plus antibiotic at recommended concentration. Allow this to grow >12hr at 37 degrees.
- (optional) Remove 1 ml of culture and add 100 ul of 100 % glycerol to make a freezer stock .
- Then transfer the remaining culture to a 15ml Corex tube on ice. Spin the cells 6K 10' 4 degrees .
- Pour off the supernatant and drain the tubes over a Kimwipe or a paper towel for 5' .
- Resuspend the pellet in 250 ul 25% sucrose solution ( vortex hard to resuspend ). While the tubes set on ice, make the lysozyme solution.
- Add 150 ul lysozyme solution to Corex tubes and then transfer to a1.5 ml Eppendorf tube ; Keep on ice for a total of15'.
- Add 500ul triton lysing solution and mix quickly by inversion. Keep on ice and do not disturb for 15' and then microfuge for 15'.
- Remove viscous pellet from bottom of the tube with a Pasteur pipette and heat supernatant at 65 degrees for 5' to ppt. proteins.
- Microfuge for 5 min. Transfer supernatant to a new tube containing 500 ul phenol ( no chloroform ), vortex 30 sec. and spin at 4 degrees for 5'.
- Transfer supernatant to a fresh tube containing 50 ul 3 M NaOAc and fill tube with absolute EtOH. Chill tube at - 20 degrees for at least 10' and microfuge 10' , drain EtOH and wipe tube walls with Kimwipe to dryness.
- Resuspend pellet in 100ul 1XTE buffer (takes only about 5' at 65 degrees). Run over a Sephadex G-50 mini column ( remember to wash the column).
- Store at -20 degrees , yield is appx. 10-20 ug. Quantitate the DNA.
* MCL plasmid preps contain lots of RNA.