User:Jarle Pahr/Cloning: Difference between revisions
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| 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 (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 || || || | | 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 | ||
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| CPEC || Fast || || ~1h (20 cycle PCR) || || | | 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 | | 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 (?) || || | | 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 | ||
|} | |} | ||
Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html | Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html |
Revision as of 10:06, 25 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/
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
Topo cloning
Polymerases
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 | ||
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 |
Polymerases: http://oregonstate.edu/instruction/bb492/lectures/DNAI.html