#3       Plasmid Mini-Preps via HomeMade  Solution  1,  2, & 3

This method for plasmid isolation is based on the Miniprep kit
(Qiagen) except  that no column is utilized in our protocol.  Instead,
the plasmid DNA is precipitated with  isopropanol.  The isolated DNA
is acceptable for restriction digests, ligation, and other  subcloning
applications.


1.      Add 1.5 ml of culture to 1.5 ml eppendorf tube.  Spin at 13,000rpm
RT centrifuge for 1  min to  pellet cells.  Decant supernatant, and
repeat up to two times if desired in same tube to collect more cells.
2.       Resuspend cells in 250 ul of #1 by vortexing (250 ul per max of 5
ml cells).  No cell clumps should be  visible in suspension.
3.      Add 250 ul of #2 and gently invert 4-6 times to mix.  DO  NOT
vortex here!  Solution should become viscous and slightly clear.  DO
NOT let lysis  proceed for more than 5 minutes or bacterial chromosome
will be extracted also (an undesirable effect)!
4.      Add 350 ul of #3 and invert tube immediately, but gently, 4-6
times to mix.  Solution should become cloudy.
5.      Centrifuge at 13,000rpm RT  for 4 minutes to pellet debris.
6.      Transfer  the supernatant to a new tube
7.      Add  750 ul of RT isopropanol to precipitate DNA  and invert 4-6x
to mix. Let tubes sit at RT for 10min to precipiate DNA. Can be placed
on ice to increase yield if desired.
8.      Centrifuge at 13,000rpm at 4C centrifuge for 10 minutes to pellet
plasmid DNA.  (RT centrifuge ok, but decrease the time to 7min)
9.      Check for a white DNA pellet. If pellet present, pour out
supernatant carefully. Don't lose pellet, which sometimes can be
"soft" and run down the side of the tube. Watch out!
10.      Washing the pellet free of salts:  Add 100 ul of  70% ethanol
briefly and pour out carefully. Not to lose the pellet. Keep an eye on
the pellet at all times.  Drain the tubes over some kimwipes to remove
excess ethanol.
11.      Air dry pellets with tubes on their sides for 20 minutes.  Ok to
dry for hours if needed.
12.     Resuspend pellet to 50ul TE usually pH 7.5-8.0. Can use 5ul for
restriction enzyme digestion and ~15ul for gel extraction.



Solutions:

 1: (50mM Tris-HCL pH 8.0, 10mM EDTA and 100ug/ml RnaseA should be
stored at 4 degrees centigrade).

2: (200mMNaOH, 1% SDS if solution is cloudy heat to 37 degrees C until
clear) – store at R. T.

3: (3.0M KoAc pH5.5) -- pH to 5.5 with Acetic Acid , store R.T.