User:Jarle Pahr/Cloning

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Line 35: Line 35:
incomplete PCR (iPCR):
incomplete PCR (iPCR):
 +
Quote from Li and Elledge 2007:
 +
 +
"Excision by the proofreading
 +
exonuclease of T4 DNA polymerase has proven to be the
 +
most reproducible and easiest to manipulate method for generating
 +
5¢ overhangs. Although much less efficient, iPCR also gives substantial
 +
stimulation of transformation. This might be sufficient for
 +
routine subcloning purposes, although there is likely to be more
 +
variability depending on the completeness of the PCR synthesis"

Revision as of 16:47, 21 February 2013

5 Tips on Vector Preparation for Gene Cloning: http://nucleicacids.bitesizebio.com/articles/cloning-tips-vector-prep/

http://www.addgene.org/plasmid_protocols/DNA_ligation/

Contents

Ligation Independent Cloning (LIC)

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332407/pdf/nar00204-0127.pdf

http://bitesizebio.com/articles/get-your-clone-90-of-the-time-with-ligation-independent-cloning/

Adding RecA may improve yield?

Gibson Assembly/SLIC

From J5 manual, "The SLIC, Gibson, CPEC and SLiCE assembly methods" (http://j5.jbei.org/j5manual/pages/22.html) :

"SLIC, Gibson, CPEC, and SLiCE are related methods that offer standardized, scarless, (largely) sequence-independent, multi-part DNA assembly".

Also: "Despite their differences in implementation, SLIC, Gibson, CPEC, and SLiCE assembly methods all start with the same starting materials and result in the same final products"

A guide to Gibson Assembly: http://www.synbio.org.uk/dna-assembly/guidetogibsonassembly.html

See also http://openwetware.org/wiki/Gibson_Assembly

One-step SLIC: http://aem.asm.org/content/78/15/5440.long

Li 2007 - Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. http://www.ncbi.nlm.nih.gov/pubmed/17293868


Enzyme free cloning for high throughput gene cloning and expression. http://www.ncbi.nlm.nih.gov/pubmed/17295099

Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. http://www.ncbi.nlm.nih.gov/pubmed/17293868

incomplete PCR (iPCR):

Quote from Li and Elledge 2007:

"Excision by the proofreading exonuclease of T4 DNA polymerase has proven to be the most reproducible and easiest to manipulate method for generating 5¢ overhangs. Although much less efficient, iPCR also gives substantial stimulation of transformation. This might be sufficient for routine subcloning purposes, although there is likely to be more variability depending on the completeness of the PCR synthesis"


USER

CPEC

GoldenGate

http://j5.jbei.org/j5manual/pages/23.html

Quote from the above: "Perhaps the most significant limitation of the Golden Gate method is that it is less sequence-independent than SLIC/Gibson/CPEC/SLiCE, in the sense that, like BioBrick assembly, the selected type IIs recognition site (e.g. BsaI) should be absent from the internal portions of all of the DNA fragments to be assembled" From the same J5 website (http://j5.jbei.org/j5manual/pages/22.html):

"Since there are no (or very few) re-amplifications of a given template sequence, PCR-derived mutations are not propagated to the same extent as one would anticipate for standard SOEing reactions. Like SLIC and Gibson assembly, CPEC is standardized, scar-less, and largely sequence-independent."

RF cloning

Molar ratio calculator: http://www.promega.com/techserv/tools/biomath/calc06.htm?origUrl=http%3a%2f%2fwww.promega.com%2fbiomath%2fcalc06.htm

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