ligation-free cloning

Maximilian Haeussler via methods%40net.bio.net (by maximilianh from gmail.com)
Mon Jun 18 06:56:45 EST 2007


>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.

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_bhagat 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
>
--



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