Use this category for questions regarding problems manipulating proteins in molecular biology applications (expression, detection, etc.)
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Hi to everybody,
I am trying to use Origami B (DE3) host cells (from Novagen) to overproduce an heterologous GST-fusion protein but I find lots of problems to grow and transform the cells. Even before being transformed, they grow very slowly (two days in the incubator at 37ºC) and the colonies have a very bad appearance. The transformation efficiency is extremely low (following all the manufacturer’s recommendations).
Does somebody know any trick to grow and transform these cells?
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- Joined: Jan 29 2004 1:51 pm
- Location: Valencia, Spain
The reason for such low growth rate is due to the fact that 4 antibiotics are need to be included in the culture. Kanamycin, tetracyclin and chloramphenicol for the host and ampillin for the plasmid (if your plasmid confer ampicllin resistance).
do the antibiotic concentration correct?
I use 100ug/ml ampicillin, 50ug/ml kanamycin, 35ug/ml chloramphenicol and 35ug/ml tetracyclin.
Under this growing condition, i can get average size colonies after at 24hours.
School of Life Science
The Chinese University of Hong Kong
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- Joined: Jul 25 2003 10:15 pm
- Location: Hong Kong
the concentration of the novagen protocol are:
kanamycin: 15 µg/ml
tetracycline: 12 µg/ml
chloramphenicol: 34 µg/mla
ampicilin:100 µg/ml (i use 60 µg/ml for expression)
Sometimes you can have problems with your plasmide (toxic gene product). In this case the cells growth is very slow. Look for a very good regulated Origami strain (Novagen)- their are some differences.
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- Joined: Feb 05 2004 9:18 am
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