mini prep procedure

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sun Apr 11 09:16:00 EST 1993


In article <C588KG.32D at ncifcrf.gov>, pnh at fcsparc6.ncifcrf.gov (Paul N Hengen) writes...
>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...
> 

I wrote:

>>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"

You wrote:

>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.

I used his words, that's why I put quotes around them.

>>(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?

True, True, I stand corrected.

>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


Paul,
	My main points were:
	1. the amp may have gone off, and
	2. freeze the cells if one has to wait before continuing with the prep.
Cheers.....bruce



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