mini prep procedure
Paul N Hengen
pnh at fcsparc6.ncifcrf.gov
Fri Apr 9 12:29:04 EST 1993
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
>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
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