Pl Hirt procedure again

Victor Levenson levenson at uic.edu
Tue Aug 12 13:47:41 EST 1997


bchjgp at leonis.nus.sg (Patil J G) wrote:

>We shall greatly appricite possible pit-falls and tips for isolation of 
>plasmid DNA (unintegrated) from cell-lines. Please post suggestions on the 
>news group or you coud mail directly to me.

>--
>**********************************
>Jawahar G Patil                  :
>Department of Biochemistry       :
>National University of Singapore :
>Singapore-119260                 :
>E-mail: bchjgp at nus.sg            : 
>Phone: (65)-7727996(O)           :
>       (65)-7742867(R)           :
>Fax:   (65)-7791453              :
>**********************************

Jawahar,

Don't even try doing Hirt isn suspension after trypsinization of
cells. For some reason this leads to VERY extensive shear no matter
how gentle you swirl the lysate - and you end up with dismal
transformation of the bugs. 

NaCl and KCl work almost the same for precipitate formation (Ihave not
tried KCl for cells in suspension, BTW, may be quicker formation of
precipitate will reduce shear), though KCL works much quicker. KCl
pellet is more brittle as compared with NaCl, so great care should be
taken not to transfer small pieces of precipitate from the tube.

Adding proteinase K to lysis solution and 2 hr incubation of lysate at
37^C before NaCl addition seem to increase approx 2-3 fold the yield
of transformants (suggested by Rob Kalejta).
After o/n incubation at 0^C, centrifugation and phenol/chloroform
extraction Ian York suggests precipitation of supernatant with Linear
Polyacrylamide (wonderful thing!), dissolving precipitate in minimal
volume and dialysis on disks against very pure water immediately
before electroporation. In my hands additional wash with 70% ethanol
gave the same efficiency as dialysed prep, but "your milage may vary".

Good luck,

Victor Levenson
Res Asst Prof
UIC
levenson at uic.edu



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