mini prep procedure
hamel at ccu.umanitoba.ca
Thu Apr 8 18:25:02 EST 1993
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.
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
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