mini prep procedure

Marianna Max drmax at casbah.acns.nwu.edu
Fri Apr 9 05:55:08 EST 1993


I did a series of minipreps where the bug growth looked good but at the end
of the procedure there was no visible pellet and no evidence of plasmid in
my gel. I was using a very reliable kit so I couldn't figure out what
was going so wrong that I would lose all the plasmid. Turns out that my
antibiotic had gone off and I was growing bugs without plasmids. Don't know
if that is your problem but it's easy to check. Amp is considered fairly
unstable both in plates and in LB media. We use Carbenicillin (sp?) and
that seems to last longer. BTW, I've had no problem with leaving the bugs
in the fridge for several days before prepping as long as the antibiotic
was fresh to start with (using bluescript vector).

Max
In article <C56uDq.23v at ccu.umanitoba.ca> hamel at ccu.umanitoba.ca (Andre Hamel) writes:


>
>What plasmid was used (vector replicon ... presumably high copy number, pUC
>based, in order to increase likelihood of "success") ... I take it this
>was for an undergrad lab .... also, what volumes of cells were used? .. 1
>mL, 10 mL, 1 Litre? Assuming ANY pellet was observed after the final
>plasmid extraction steps:
>was ANYTHING seen on an appropriate agarose gel (say 1% in 1x TBE, run
>at, say about 75 Volts/no more than 100 mAmperes for no more than an
>hour for a 10 cm long mini gel) after ethidium bromide/UV flourescence? Did
>you observe any smearing high or low mol. wt.? How much of the plasmid
>preps were loaded on the gels? Regarding the LiCl ppt'n .. done on ice for
>at least 1/2 hour, using about 3- 4 Molar final conc.? I'm asuming you
>followed the Maniatis Second edition 1989 manual .. pg. 13.72 refers to
>reliable LiCl ppt'n to remove much (not all) RNA ... also, was adequate
>scaling of reagent volumes used?
>
>Most higher copy number plasmids shouldn't get degraded whilst in the
>cells at 4oC over weekend, although yields do tend to be better when cells
>used "fresh", unless the plasmids have sequences/inserts expressed in the
>bacteria yielding harmful effect(or)s.
>
>regards
>
>  
>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.
>
>



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