Luciferase plasmid yields
malexeyev at biost1.thi.tmc.edu
Tue Jul 16 15:44:07 EST 1996
In article <whc-1607962100400001 at macr3-17.welc.cam.ac.uk>,
whc at mole.bio.cam.ac.uk (W.H. Colledge) wrote:
> We are having difficulties obtaining good yields of a CMVluciferase
> plasmid grown in DH5 alpha. Typically we only obtain about 50ug from 300ml
> TB cultures. Has anyone had similar problems? Is breakthrough luciferse
> expression toxic to E.Coli? Many thanks.
> Bill Colledge
> whc at mole.bio.cam.ac.uk
What protocol for plasmid isolation do you use? In my hands, TG1 would
yield up to 12-15 milligrams of pGL2 control (SV40 promoter + luciferase)
from 1 liter of TG1 after double banding in CsCl gradient. pCDNA3 with
luciferase cloned in it gives similar yield (CMV promoter).
My suggestions: try different strain (recA+)
try different protocol (especially if you are using a kit) and
see what your yields are going to be.
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