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

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(Gibson Assembly/SLIC)
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|[http://wiki.bu.edu/ece-cidar/index.php/MoClo MoClo]|| || || || || ||

Revision as of 09:48, 15 March 2013

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


Blunt end ligation:



Conventional (restriction-ligation) cloning






Ligation Independent Cloning (LIC)



Adding RecA may improve yield?

If no colonies are achieved, try "quick and dirty" cloning: http://network.nature.com/groups/natureprotocols/forum/topics/1284

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

NEBs NEBuilder software for Gibson assembly can be used to design primers for Gibson assembly and SLIC: http://nebuilder.neb.com/

In the NEBuilder, the overlaps are designed to achieve a minimum Tm value of 48°C.

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


RF cloning can be used to insert a sequence into a plasmid without removing any of the original plasmid sequence, or to replace a portion of a plasmid with a new sequence. RF cloning is accomplished using two PCR steps. In the first PCR, the sequence to be inserted is amplified (or synthesized) to give a product with the insert sequence flanked by sequences homologous to the plasmid. In the second PCR, the product of the first step is used as a "megaprimer" To insert a sequence into a plasmid without removing any of the original plasmid sequence, the plasmid-binding regions of the megaprimer should bind to the plasmid directly adjacent of each other, as any sequence in the plasmid between the two primer binding sites will be lost. To replace a portion of a plasmid, the plasmid-binding flanking sequences of the megaprimer must be designed to bind such that they flank the area to be replaced. If RF cloning is used to replace a portion of a plasmid, the new sequence should be about as long as the sequence it is replacing.

Topo cloning


Cloning methods

Method Summary Advantages Disadvantages Time (for assembly from prepared vector and insert) Experiences Reference
Conventional (restriction enzymes) Restriction digestion and ligation Predictable Requires suitable restriction sites in vector From 3 h to overnight
Blunt-end ligation Ligation of blunt-ended fragments Flexible, does not require specific restriction sites. 10-100x less efficients than cohesive-end ligation. Requires screening of colonies (insertion is non-directional). From 3 h to overnight https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/06/15/cloning-strategies-part-3-blunt-end-cloning?c=EU
SLIC (one-step) Annealing of fragments with Homologous overlaps 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) Annealing of fragments with Homologous overlaps 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

http://www.nature.com/protocolexchange/protocols/167#/procedure http://www.nature.com/protocolexchange/protocols/170#/procedure

iPCR Annealing of vector and incomplete PCR fragments with homologous overlap 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 Circularization of linearized vector and insert with homologous overlap Fast Requires linearized vector ~1h (20 cycle PCR) http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006441
Gibson Assembly Annealing of fragments with homologous overlaps. 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 Addition or substitution of a sequence in a plasmid by PCR 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 Another name for PIPE/iPCR http://link.springer.com/article/10.1007%2Fs10969-006-9014-z
Sucessive Hybridization Assembly (SHA) In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

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

MASTER ligation: http://nar.oxfordjournals.org/content/early/2013/02/26/nar.gkt122.full

Use of Phosphatases: