mini prep procedure
cytogen at chmeds.ac.nz
Fri Apr 9 18:37:21 EST 1993
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
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
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
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.
Martin A Kennedy (E-mail = cytogen at chmeds.ac.nz)
Cytogenetic and Molecular Oncology Unit
Christchurch School of Medicine
Christchurch, New Zealand
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