I ordered L4440 plasmid (DH5alpha growth strain). I was unable to extract plasmid using miniprep kit. My growth medium is LB+amp. Any advice is welcome. I suspect that I may have lost the plasmid in the growth strain. Is there any way how I can recover it?
A summary of what you did might help everyone.
How much plasmid was used to transform which DH5a exactly and did you plate the transformation on LB Amp plates? What was the Amp concentration in the agar and how old were the plates? Can you calculate the CFU per ug of DNA you used to transform, as that may help diagnose a transformation problem. Did you get a couple colonies or a near lawn?
If you transformed with a low amount of plasmid (1 ng or less) and picked a single colony for a liquid mini culture (with what conc. of amp?) grown overnight (15 - 18 hours), then you have to distinguish between the bacteria losing the plasmid and the miniprep kit not working for some reason. L4440 has a pBR322 origin, so it's not a very high copy number vector compared to pUC ori vectors (20 copies per cell vs. 500+ copies, respectively). How much liquid culture did you prep and how much did you analyze?
That might be a good place to start.
Hi Feysel,
I suggest to plate first your DH5alpha with different concentrations of Amp overnight, if you get good colonies then inoculate in liquid media with the respective Amp concentration, if you don"t get good colonies try to incubate for longer time.
Alternatively, check the Miniprep kit protocol, is the buffers provided ready to use? do in copies your extraction, as the capacity of a spin column is 20ug. After elution run an agarose gel alongside a DNA Ladder to check the size of your plasmid and eventually to estimate the concentration as well.
Good luck
Hi Feysel,
When your plate cultures are oké then I would suggest that to check you mini mix after overnight incubation before extracting the construct out of your miniprep. Look at the optical density (600nm) of your culture and only when the value is high enough extract otherwise let it grow for a longer time. I would also suggest extracting a control if you don't thrust the quality of your the mini prep buffers.
Good luck
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