SLICE -- Seamless Ligation Cloning Extract... or, using lambda phage homologous recombination in-vitro

[Nathan McCorkle]

this is a pretty decent method, turns out the folks that did this are
right up the street from me... maybe I can talk to them

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3333860/pdf/gkr1288.pdf

Published online 12 January 2012 Nucleic Acids Research, 2012, Vol.
40, No. 8 e55
SLiCE: a novel bacterial cell extract-based DNA cloning method
Yongwei Zhang*, Uwe Werling and Winfried Edelmann*
Department of Cell Biology, Albert Einstein College of Medicine,
Bronx, NY 10461, USA
Received July 28, 2011; Revised December 13, 2011; Accepted December 15, 2011


Here's to homologous recombination!



--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Nathan McCorkle]

P.S. this seems like something Cathal would be interested in... since
it seems like nothing more than a crude cell lysate/extract

[Cathal Garvey]

oooo! I love crude things! :d
Thanks! :)

www.indiebiotech.com
twitter.com/onetruecathal
joindiaspora.com/u/cathalgarvey
PGP Public Key: http://bit.ly/CathalGKey

[yamwa]

Did anyone try out this method? I got the PPY strain from Einstein but don't have luck with it :/

[Nathan McCorkle]

On Thu, Sep 12, 2013 at 2:54 PM, yamwa <yam...@gmail.com> wrote:
> Did anyone try out this method? I got the PPY strain from Einstein but don't
> have luck with it :/

Didn't have luck in what sense? How long were your homologous flanking regions?

[Andrew]

I too got this strain and no luck, but I'm no pro.  Anyone else have trouble or success?  A recent article on synthetic assembly methods stated that of survey respondents, around 10%, or 13 respondents, used SLICE.  I might try CPEC instead, and I really want ABI-REC to work, but so far no luck...

[Rob C]

Our lab uses the PPY strain (a generous gift from Einstein U) for our cloning needs. We exactly follow their prescribed protocols to prep the extract, buffer, etc.

This method is a true gem. Side by side with Gibson, it works at least as well and often better. For basic insertion of genes into vectors we obtain lawns of cells (too many colonies sometimes), and all tested colonies are colony PCR positive and sequencing comes back as intended.

Economically it is very difficult to argue using Gibson over PPY-based SLiCE cloning.

[Mega [Andreas Stuermer]]

What if your plasmid contains repeats? Will there be unwanted recombination?

[Rob C]

Good question, I haven't worked with repeats with slice.

I suspect it'll be heavily dependent on repeat size, number of repeats, content, linkers, where the repeats are in the plasmid relative to place of intended insertion and homology ends, etc. Hard to speculate really...only one way to know for sure. ;)

[Cihan Aydin]

An hack to SLiCE that I found very useful in the lab...

https://wiki.london.hackspace.org.uk/view/SPLiCE

Basically you add like %3 PEG 8k to the reaction mixture and boom it works much better.

I have used SLiCE and SPLiCE for assemblies up to 7 parts and around 2kb long for gene synthesis and mostly codon optimizing genes for expression in other organisms. The most important part is the overlap regions in my experience, where they should be basically unique with respect to one another.

2017 Motohashi protocol on Springer is overall a good starting point on optimizing your reaction. - http://link.springer.com/protocol/10.1007%2F978-1-4939-6472-7_23

Good luck!

Cihan Aydin, Ph.D.

Linkedin: www.linkedin.com/in/cihanaydinphd
RG: www.researchgate.net/profile/Cihan_Aydin

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[Michael Crone]

Why not just use CPEC instead of requiring a separate enzyme mix just for cloning? Phusion works absolute wonders and I've never had any problem creating large plasmids.

[Rob C]

Probably because it requires an extra PCR reaction. 

The separate SLiCE "enzyme mix" you refer to costs nothing, and with about 2 hrs of prep work one can make enough for 10,000+ reactions. PCRs are not free, often require troubleshooting and can be time consuming. The fewer PCRs one has to do the better in my book. 

Granted not all cloning methods work equally well for all situations, but for standard insertion of an insert into a linearized vector it's pretty hard to beat the convenience, cost, efficiency and accuracy of recombinase-expressed cell extract SLICE methods. I suspect those making $$ on Gibson reagents are working hard to ensure these methods don't become commercialized.