Blunt end cloning

mnr mnr via methods%40net.bio.net (by mnr475 from gmail.com)
Wed Jul 6 18:57:36 EST 2011


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 <novalidaddress from nurfuerspam.de> 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 <mnr... from gmail.com> 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
>
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