Dominic-Luc Webb molmed
domweb at mbox.ki.se
Tue Aug 14 14:51:31 EST 2001
On 14 Aug 2001, Dr. Peter Gegenheimer wrote:
> We have used, very successfully, a clone which lacked the T7 terminator.
> I kept telling my student that the terminator was needed, and he kept
> showing me that it wasn't...
Studier 1986 and 1990 (refs in Sambrook I have read recently)
indicate that there could also be an RNase III rather than a
T7 terminator. Perhaps the plasmid your student has contains
this instead? Or perhaps there is some other stem loop
Also, regarding the pET vector, as near as I can tell
from the maps offered by Novagen, it does indeed
appear that the target gene is in same orientation
as amp resistance....
Our plasmid is built from the pRcCMV, a decendant of the pBR322,
as the pET plasmids are. However, Invitrogen, the supplier of
pRcCMV, indicates the opposite orientation for amp resistance
and target gene, assuming I am reading the map correctly and
taking into account the pBR322 numbering system. As near as we
can tell, our plasmid is essentially the same as pET in other
regards, except in lacking the T7 terminator. I also note that
under the same conditions we have used, 3 hours 1 mM IPTG
treatment at 37 degrees C, Novagen is apparently able to get
quite good expression of EGFP, our present target gene. They
show a stained gel of a similar plasmid expressing EGFP in
the BL21(DE3) cells we are using.
Our antibiotic protocol is pretty stringent, 200 ug/ml
at start of induction, and another addition of amp after
one hour, just to be sure.
Since the medium, etc used if of interest, I am curious if
the details of your IPTG induction are published... medium
Dominic-Luc Webb, doktorand
Department of Molecular Medicine
Endocrinology and Diabetes Unit
Rolf Luft Center for Diabetes Research
Karolinska Hospital L3
Internet Email: dominic at enk.ks.se
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