ligation-free cloning

chhoefer via methods%40net.bio.net (by chhoefer from gmail.com)
Mon Jun 18 16:51:31 EST 2007


On Jun 18, 9:05 pm, ChenHA <h... from freeuk.com> wrote:
> Maximilian Haeussler wrote:
> >> This is just a reminder for u whether u get any further information
> > regarding PCR
> >> ligation-free cloning
>
> > not completely but close: the best method is enzyme-free cloning,
> > (optional: in a combination with exonuclease recession-treated
> > vectors, in case that your vector is too big for a PCR, see my last
> > mail), the original reference for this is "Enzyme-free cloning: a
> > rapid method to clone PCR products independent of vector restriction
> > enzyme sites" , Tillett and Neilan NAR 1999.
> > I've been too busy to try this myself but a recent publication from
> > 2006 [1] is saying that this system is (they call it LIC-PCR, tested
> > on several hundred clones)
> > a) cheaper
> > b) faster and
> > c) as reliable as the highly expensive gateway cloning enzymes from
> >invitrogen
>
> > They plan to publish the exact protocol that they used with their
> > pipetting machines.
>
> > These "new" cloning protocols seem to be quite an improvement over
> > restriction-enzyme-based-cloning, given that they allow automation,
> > are cheaper and the same success rate as recombination-based (e.g.
> > gateway) cloning systems.
>
> I was curious and had a look at it, and your summary seems a bit off.
> LIC-PCR is different from the Tillet and Neilan one (i.e. EFC, and EFC
> works better than LIC-PCR of the 24 constructs tested, not hundreds),
> and LIC-PCR is only cheaper compared to Gateway and Infusion. Certainly
> I don't think EFC is cheaper when compared to normal cloning methods
> (the original paper admits that it is more expensive).
>
> > cheers,
> > max
>
> > [1]
> > Alzari PM, et al, Implementation of semi-automated cloning and
> > prokaryotic expression screening: the impact of SPINE.
> > Acta Crystallogr D Biol Crystallogr. 2006 PMID: 17001088
>
> > -------
>
> > On 18/06/07, Neeru Bhagat <neeru_bha... from yahoo.com> wrote:
> >> Dear Max
>
> >> This is just a reminder for u whether u get any further information
> >> regarding PCR ligation-free cloning
>
> >> Thanks
> >> Neeru
>
> > --

Hi,

   Gateway cloning is just a few dollars per reaction for a clone you
are sure to get.   Do you really think this
is a lot of money for something that otherwise might take weeks,
months or forever to produce?

Christoph



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