ligation-free cloning

Maximilian Haeussler via methods%40net.bio.net (by maximilianh from gmail.com)
Tue Jun 19 07:29:42 EST 2007


> 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).

Sorry, I stand corrected and admit being overenthusiastic. LIC (=T4
recessed ends) is the one that has been used on 263 clones, EFC
(=overlaps created by 2 pairs of different primers) has "only" been
tested on 24 constructs in this benchmark.

Prices of primers have changed a lot since the paper was written.
EFC is cheaper than methods with the same efficiency (gateway or
in-fusion). "Normal cloning" (=recessed ends created by restriction
enzymes) is of course the cheapest method but less effective.

Christoph write
>  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?

The BP/LR clonase enzyme mixes cost around 15 EUR per reaction. the
whole protocol takes several overnight steps: As far as I know (not
sure, don't want to be overenthusiastic again :-) according to this
protocol http://crfb.univ-mrs.fr/ciona/lemaire/download/Detailed_Gateway_protocol.pdf,
it takes 6 days from your PCR product to a miniprep of your plasmid
using gateway, whereas it seems to take only a couple of hours for the
hybridization + bacterial overnight culture using EFC/LIC.
I admit that 15 euros per clone is not that much money, even more
since you can reduce this cost by reducing the volume of the gateway
reactions. But why rely on invitrogen if there is an alternative that
is as effective?

BTW: Aren't you working at Invitrogen, Christoph?

cheers,
Max



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