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ligation-free cloning

ligation-free cloning - Protocols and Methods Forum

ligation-free cloning - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-18-2007, 11:56 AM
Maximilian Haeussler
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Default ligation-free cloning



>This is just a reminder for u whether u get any further information
regarding PCR

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 <[Only registered users see links. ]> wrote:
--

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  #2  
Old 06-18-2007, 07:05 PM
ChenHA
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Default ligation-free cloning

Maximilian Haeussler wrote:


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).




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  #3  
Old 06-18-2007, 09:51 PM
chhoefer
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Default ligation-free cloning

On Jun 18, 9:05 pm, ChenHA <[Only registered users see links. ]> wrote:

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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  #4  
Old 06-19-2007, 12:26 AM
peter
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Default ligation-free cloning

On Jun 18, 5:51 pm, chhoefer <[Only registered users see links. ]> wrote:

LIC works well.... you may want to look NEB USER cloning....
my2c

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  #5  
Old 06-19-2007, 05:07 AM
DK
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Default ligation-free cloning

In article <[Only registered users see links. ].net> , "Maximilian Haeussler" <[Only registered users see links. ]> wrote:

I am using with great success what I call "QuickChange cloning".
In brief: 1) you PCR out a gene of interest with primers that have 20-30 bp
extra "arms" on both 5' and 3' ends that are designed to anneal to the
plasmid the gene is to be cloned into; 2) you use that PCR product
in place of primers in what is essentially a "QuickChange protocol"
(e.g., whole plasmid PCR); 3) treat the product with DpnI to degrade
parent methylated plasmid and transform.

I've originally come up with the method on my own, purely out of
necessity to clone in frame on both N- and C-terminus without cloning
artifacts into a stupid vector without good sites. Works great.
Only later, after my boss suggested that we publish the method as a brief
note in Biotechniques, I have discovered that essentially the same
method was already published three times (the two later papers don't
site the original 2000 paper in Biotechniques!) and the same thing
is used in Stratagene's "Genemorph EZClone" kit. (Hehe, I *knew*
it was a trivial idea).

In case anything is interested, Pubmed search numbers are:

10723563
12449380
16480772

Unlike others, I had an option to use new Stratagene's "Pfu Fusion"
polymerase and find that it increases success rate in comparison
to their regular Pfu Ultra.

DK
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  #6  
Old 06-19-2007, 02:30 PM
ChenHA
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Default ligation-free cloning

chhoefer wrote:


Not sure who this is directed to, but I haven't come across anything
that takes weeks, months or forever to clone.

People can use whatever kit they like as long as it works for them. I
think however relying on kits tends to degrade the skill factor. I have
made many constructs that can't be done by using kits, and those who
rely on kits alone are limited in what they can do. Normal cloning
method is simple if you know what to do, unfortunately many people
simply don't know how to do it properly.

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  #7  
Old 06-19-2007, 03:41 PM
peter
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Default ligation-free cloning

On Jun 19, 10:30 am, ChenHA <[Only registered users see links. ]> wrote:

Amen to that....can't agree more with you
Peter

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  #8  
Old 06-20-2007, 09:34 AM
Duncan Clark
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Default ligation-free cloning

Historians believe that in newspost
<1182203491.880109.150560@c77g2000hse.googlegroups .com> on Mon, 18 Jun
2007, chhoefer <[Only registered users see links. ]> penned the following literary
masterpiece:

Not all scientists are in the West. A few dollars can be a lot of money
elsewhere. It is all relative.

Duncan
--
I love deadlines. I especially like the whooshing noise they make as
they go flying by.

Duncan Clark
GeneSys Ltd.
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  #9  
Old 12-29-2011, 03:06 AM
Pipette Filler
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Default Re: ligation-free cloning

Dear Chen,

I am learning to do cloning using the ligation method here. Unfortunately I am one of the those who need to learn how to do cloning well.

I have done PCR of my insert and grew my plasmid, but then I always have problems getting colonies. Either I do not do gel extraction and risk getting billions of colonies or I do gel extraction of my insert and vectors and getting no clony. =(

Would you please give me some tips on your cloning method?

Thank you.
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  #10  
Old 01-04-2012, 01:09 PM
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Default Re: ligation-free cloning

hey ,
thanks for providing such an information
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