Frosty Loechel pulm012 at uabdpo.dpo.uab.edu
Sun Sep 18 08:23:18 EST 1994

In Article <356q2l$cq2 at mserv1.dl.ac.uk>, mbjpl at seqnet.dl.ac.uk wrote:
>Dear all,
>We are having trouble with Invitrogens Eukaryotic expression
>vector pcDNA3. 

 Another strange phenomena was that two distinct sises
>of colonies were seen and their ability to grow on the plate
>correlated with the growth in liquid culture with the small
>colonies taking about 24hrs to grow to stationary phase. 

Has anyone got any ideas
>what is going on or have any advice.  

pcDNA3 has a cryptic E. coli promoter. It will often express your gene in E.
coli, when your gene is in the correct orientation for eukaryotic
expression. The level of toxicity to E coli will depend on your particular
gene product, and on efficiency of translation initiation. I solved this
problem for one of my constructs by sticking a prokaryotic transcription
terminator at the Hind III site, upstream of my gene. Eukaryotic expression
was not altered, but expression in E coli was reduced >99%.


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