mini prep procedure

[Martin Kennedy]

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.

Dis you streak out the primary transformants on an Amp plate before growing the
overnights?  While most of us don't bother, you have to rememeber several
things:

1.  There is an enormous background of viable (but amp sensitive) host cells on
the primary transformation plates - these cells are not killed by the
ampicillin, as evidenced by the satellite colonies you often get around
resistant colonies.

2.  Ampicillin is actively degraded by any cells bearing the B-lactamase gene
(ie any transformed cells), so that an inoculum consisting of 

3.  Ampicillin rapidly loses its potency - amp plates, or LB-amp, stored in the
fridge for only a week or two, give very poor selection.

My guess is that your overnight cultures have been over-run with sensitive
cells, making it appear that you have lost your plasmid.  Cells bearing
plasmids tend to grow more slowly than plasmid-less cells in the absence of
selection.  This might seem a trivial suggestion, but I've seen it happen more 
than once, and the solution to the problem has been to be more stringent in 
the purification and selection of transformants.
-- 
Cheers,

Martin

Martin A Kennedy (E-mail = cytogen at chmeds.ac.nz)    
Cytogenetic and Molecular Oncology Unit               
Christchurch School of Medicine                       
Christchurch, New Zealand