Difference between revisions of "User:Jarle Pahr/Cloning"

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(Cloning methods)
(Cloning methods)
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|iPCR ||  || No enzymes required (except for PCR) || Low efficiency || ~ 1 h ||
|iPCR ||  || No enzymes required (except for PCR) || Low efficiency || ~ 1 h ||http://www.nature.com/protocolexchange/protocols/171#/main
|PIPE|| ||  No enzymes required (except for PCR)|| || ||http://www.ncbi.nlm.nih.gov/pubmed/18988020
|PIPE|| ||  No enzymes required (except for PCR)|| || ||http://www.ncbi.nlm.nih.gov/pubmed/18988020

Revision as of 09:28, 25 February 2013

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


Ligation Independent Cloning (LIC)



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"





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

Topo cloning


Cloning methods

Method Advantages Disadvantages Time (for assembly from prepared vector and insert) Experiences Reference
Conventional (restriction enzymes) Predictable Requires suitable restriction sites in vector From 3 h to overnight
SLIC (one-step) Fast Insert should be at least 250 bp. More secondary structures might be present when incubating at room temperature, compared to two-step SLIC and Gibson assembly. ~30 min http://aem.asm.org/content/early/2012/05/13/AEM.00844-12
SLIC (two-step) Fast. Assembly is carried out at higher temperature than one-step SLIC, meaning secondary structures less problematic? Slower than one-step SLIC http://www.nature.com/nmeth/journal/v4/n3/abs/nmeth1010.html


iPCR No enzymes required (except for PCR) Low efficiency ~ 1 h http://www.nature.com/protocolexchange/protocols/171#/main
PIPE No enzymes required (except for PCR) http://www.ncbi.nlm.nih.gov/pubmed/18988020
CPEC Fast Requires linearized vector ~1h (20 cycle PCR) http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006441
Gibson Assembly Relatively fast. Assembly is carried out at higher temperature than SLIC, meaning secondary structures less problematic? More enzymes required than for SLIC. http://j5.jbei.org/j5manual/pages/79.html
RF cloning Can use uncut vector as starting material. Can be used to either replace to replace a sequence in the original plasmid with a new one, or insert a new sequence without removing any of the original plasmid sequence. If the insert shall replace a sequence, the insert and the sequence to be replaced must be of about the same size. (Anectodal. Source?) ~3h (?) http://www.sciencedirect.com/science/article/pii/S0165022X06000029
Enzyme-free cloning http://link.springer.com/article/10.1007%2Fs10969-006-9014-z

Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html