mini prep procedure

[Paul N Hengen]

In article <9APR199307451104 at aardvark.ucs.uoknor.edu>
  broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:

>In article <9304081946.AA26174 at mainrt.luther.uni.edu>
  krauske at MAINRT.LUTHER.UNI.EDU (Kevin Kraus) writes...

>>	we are wondering if anyone has a solution to the following problem.
>>three different plasmids were transformed into dh5 alpha competent cells.  
>>they were then grown on amp lb plates to select.  they were then grown in 
>>amp lb broth overnight and then stored in the fridge.  over the next week,
>>minipreps were attempted by approximately 40 students using alkaline lysis,
>>and licl and ethanol precipitation, the results were null.  is there a 
>>possibility that the plasmids are degrading while in the fridge, or during
>>the prep?  all solutions appear to be fine.  any reply would be greatly
>>appreciated.
>
>An alternative possiblity is that the amp was old or left out.  Check it out.
>If you collect the cells after growth (by cntfg), pour off the super, and
>then FREEZE the pellets, you'll eliminate any chance of "degradin while
>in the fridge"

I am of the opinion that the plasmids are not degraded, but rather the cells
continue to metabolize and produce EPS, toughening the cell walls, etc. when
left at 4 C. I think this because plasmid DNA can be extracted from non-viable
ancient cells by the mini-prep method I posted previously. Of course the best
practice would be to simply regrow the cells in fresh antibiotic broth. If in
a pinch however, you could do the mini-prep and transform competent cells to
recover your clones.

>(also will help with lysis because the ice crystals will
>partially disrupt the cells upon thawing).
                                  ^^^^^^^
Didn't you mean ice crystals _forming_ will disrupt the cells?

I would think it best to add the lysis buffer first, wait 5 minutes,
and then freeze them as lysate. Better yet...do the mini-prep up until
the ETOH ppt step and then place at -20 C. It's not that much more time spent.

Paul N. Hengen
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, Maryland 21702-1201 USA