Hi Wo. It is a outer membrane protein, toxicity is probably at expression level. I get inserts with random deletion that I can not use for functional assays.
Before I try other expression systems, I want to try other E. coli vectors first. Am I going in the right direction? On Wed, Jul 6, 2011 at 10:49 PM, WS <[email protected]> wrote: > Hi Matt, > > does that mean that you don't get any clone with an insert at all or > "just" no clone that has a correct insert (full length, no deletions, > no frameshifts etc.)? > pUC18 is promoterless, right? > > As you suspect inherent toxicity, do you have any idea what kind of > toxicity it is? Will you need that toxic element or may you make it > "innocent" by some sort of AA substitution / motif deletion? Can you > possibly switch to another host like some sort of phage, YAC, (really > safe!!!) virus etc? > > Wo > > > > > On Jul 6, 7:23 pm, mnr mnr <[email protected]> wrote: > > Hi all. > > > > I have tried cloning a potential toxic gene about 1.2 kbp into E.coli > with > > T7 promoter. After two months, I am still not successful. I am now > plannig > > to do cloning into pUC18 vector before subcloning into few other vectors > > with different promoters. I have limited available restriction sites so > that > > I don't have to redesign PCR primers. Is it feasible to do blunt-end > cloning > > with Phusion polymerase into non-dephosphorylated vectors? Will I only be > > getting empty vectors after transformations? > > > > Thank you. > > > > Matt > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
