User:Jarle Pahr/Cloning
5 Tips on Vector Preparation for Gene Cloning: http://nucleicacids.bitesizebio.com/articles/cloning-tips-vector-prep/
http://www.protocol-online.org/biology-forums/cloning.html
Recombineering: http://www.biotec.tu-dresden.de/research/stewart/group-page/recombineering-guide.html
Inverse Fusion PCR cloning: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035407
Blunt end ligation:
In-Fusion Cloning primer design tool: http://bioinfo.clontech.com/infusion/convertPcrPrimersInit.do
Reviews
http://www.ncbi.nlm.nih.gov/pubmed/15489321
Conventional (restriction-ligation) cloning
http://openwetware.org/wiki/IGEM:Harvard/2008/Lab_Notebooks/GenProtocols#Protocol_Notes
http://www.addgene.org/plasmid_protocols/DNA_ligation/
http://www.addgene.org/tools/protocols/cloningprotocol/
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/
http://bitesizebio.com/articles/ligation-independent-cloning-primer-design/
Adding RecA may improve yield?
NE-LIC: http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057943
http://noxtoolbox.autresmondes.eu/LICgenerator1beta/Licfaq.html
http://noxtoolbox.autresmondes.eu/LICgenerator1beta/
If no colonies are achieved, try "quick and dirty" cloning:
http://network.nature.com/groups/natureprotocols/forum/topics/1284
http://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/LIC-cloning.pdf
Gibson Assembly
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.
SLIC
Enzyme free cloning for high throughput gene cloning and expression. http://www.ncbi.nlm.nih.gov/pubmed/17295099
incomplete PCR (iPCR):
Articles related to LIC and SLIC:
Author(s)/year Year | Comment | Reference |
---|---|---|
Aslandis & Jong 1990 | Introduction of LIC | http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332407/ |
Hsiao 1993 | ||
C Aslanidis, P J de Jong and G Schmitz 1994 | Study on minimal length requirement of SLIC overlaps. Lowest overlap length yielding transformants was 10nt. | http://genome.cshlp.org/content/4/3/172.full.pdf |
Lisa D Cabrita, Weiwen Dai and Stephen P Bottomley 2005 | A family of E. coli expression vectors for laboratory scale and high throughput soluble protein production | http://www.biomedcentral.com/1472-6750/6/12 |
Li & Elledge 2007 | "Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC." | http://www.ncbi.nlm.nih.gov/pubmed/17293868 |
2012 | One-step SLIC | http://aem.asm.org/content/78/15/5440.long |
According to Olieric 2010 (Supplementary material), "The critical parameter for success is the sequence of the overlap; some constructs work amazingly well, while others do not".
The cause could plausibly be stable secondary structures?
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"
Protocols for LIC/SLIC and related methods:
http://www.biomedcentral.com/content/supplementary/1472-6750-10-56-s1.doc
Author(s) | Hsiao 1993 | Aslandis & Jong 1990 | |||
---|---|---|---|---|---|
Method: | With RecA | Without RecA | With iPCR | Exonuclease III | LIC |
Step 1 | |||||
Step 2 | |||||
Step 3 |
SEFC/Co-transformation cloning
Mix 5 μl of purified PCR product (100–300 ng) and 1 μl (50 ng) of the appropriately linearized vector, having 15+ bp homologous overlap, before transformation.
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
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.
http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid
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 | |
LIC | Annealing of fragments with standardized homologous overlaps lackign one nucleotide | Can generate specific overhangs by exonuclease incubation in presence of a single dNTP. Standardized overlaps gives higher predictability(?). | Requires specific sequence in vector | ? | Aslanidis & Jong 1990 | |
SLIC (one-step) | Annealing of fragments with varying 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) | 1,2,3,4,5 | ||||
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 | 1,2,3 | ||||
Sucessive Hybridization Assembly (SHA) | In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization | |||||
MoClo | ||||||
Simple Cloning | PCR Assembly of insert and vector with homologous overlaps. | |||||
Seamless Enzyme-Free Cloning (SEFC)/co-transformation cloning | Co-transformation of linear insert and vector fragments | Simple, one-step co-transformation of insert and vector. No exonuclease treatment. | Requires high-efficiency competent cells (>1 × 108 colony-forming units [cfu]/μg DNA) | 1 2 | ||
Transfer PCR | Application of RF-cloning | http://www.ncbi.nlm.nih.gov/pubmed/21515384 |
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:
http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=2&t=29738
Older articles
Exonuclease III induced ligase-free directional subcloning of PCR products: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC310601/pdf/nar00072-0249.pdf
Rapid cloning by homologous recombination in vivo: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331480/pdf/nar00064-0257.pdf