ligation-free cloning

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Mon Jun 18 14:05:04 EST 2007


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_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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