help

kresten no-sp at m.org
Wed Jun 13 03:53:41 EST 2001


In general it is important to use a fresh colony of coli for expression.
Also, if you use overnight cultures for dilution into your larger flasks
this may give differences related to how long the overnight cultures have
grown. If the cells do not like to (over)express your protein, there may be
selection on non/low producers even in the presence of plasmid selection.

I suggest that you make a fresh plate of your coli strain from the freezer.
Instead of making overnight cultures I suggest that you take a large colony
from this fresh plate and inoculate into a small pre-culture (e.g. 10-20 ml)
in the morning. Then around noon dilute this preculture and dilute into your
final flask (e.g. 250 ml) and follow until desired OD then induce (if that
is what you do). This should give reproducible (and highest?) production
levels, although it in some cases may also give a long working day.

good luck
Kresten



"yangxinxiu" <yangxinxiu at yahoo.com.cn> wrote in message
news:20010613083057.13382.qmail at ww02.jatek.com...
> I use pet22b to express my protein,a month later,i can see some difference
> from sds-page between uninduced and after-induced whole cell
extraction.but
> now,i can't see the same result in the same conditions.How can I find the
> reason for this?does the Ecoli have some
> genetic difference?
>
>
>
> <http://www.biowww.net/forum/read.php?f=1&i=2430&t=2430>
>





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