Blunt end cloning

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Wed Jul 6 16:49:49 EST 2011


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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