User:Jarle Pahr/Ligation

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Notes on DNA ligation:

http://bitesizebio.com/articles/the-basics-how-does-dna-ligation-work/


https://eu.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/04/11/cohesive-end-cloning


According to above, "total concentration of DNA in the ligation reaction should be less than 10 µg/mL" (= 10 ng/uL). For 20 uL reaction volume, this equals 200 ng.

Most protocol seem to suggest between 25-200 ng vector.


Taq DNA ligase is NOT a substitute for T4 DNA ligase.

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


http://labs.fhcrc.org/fero/Protocols/Cloning.html

iGEM protocol: http://partsregistry.org/Help:Protocols/Ligation

Protocol, oomocyteworld: http://oomyceteworld.net/protocols/LigationCloning.pdf

Protocol, jkirkbrown.com: http://www.jkirkbrown.com/pdfs/Standard%20Ligation%20Protocol.pdf

Protocol, NEB T4 DNA ligase: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

Protocol,D.W Andrews lab: http://www.dwalab.ca/common/pdf/dna4.pdf

http://www.bio.davidson.edu/courses/molbio/protocols/ligation.html

Protocol, Biologicalworld: http://biologicalworld.com/ligations.htm

Protocol, Ivaan.com: http://ivaan.com/protocols/131.html

Protocol, Addgene: http://www.addgene.org/plasmid_protocols/DNA_ligation/

Discussions:

http://network.nature.com/groups/natureprotocols/forum/topics/1284

http://www.methods.info/Methods/RNA_DNA/ligation_simple.html

Comparison of protocols:


Protocol source Vector amount Insert:vector ratio Incubation Reference
Biologicalworld 200ng - 14-16 C ON http://biologicalworld.com/ligations.htm
iGEM 25 ng 1:1 16 C, 30 min http://partsregistry.org/Help:Protocols/Ligation
oomyceteworld 200-400 ng 2:1 - 6:1(1:1 after CIP treatment) "Dictated by convenience". 4-20 C for 4 to 24 h. 4-12 C recommended for sticky ends. 16 C recommended for maximum efficiency. http://oomyceteworld.net/protocols/LigationCloning.pdf
NEB 50 ng (3kb vecotr) 3:1 16 C ON or Room temp 10 min https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202
Addgene25 ng3:1RT 2 h or 4C ONhttp://www.addgene.org/plasmid_protocols/DNA_ligation/


Personal experience/recommendations:

Always do a backbone-only ligation control. If the insert has been PCR-amplified using a template with same selection marker as the desired construct, also do a insert-only ligation control to guard against plasmid contamination from the template.

Starting protocol:

~100 ng equivalents backbone DNA. 3:1 - 5:1 molar ratio insert/backbone 2 uL T4 DNA ligase buffer 0.5 uL T4 DNA ligase H2O to 20 uL

Incubate at 16 C for 3 h or overnight.


Dephosporylation:

Phosphateses such as Calf Intestinal Phosphatase can be used to dephosporylate DNA.


http://www.nirs.go.jp/report/nene/h13/03/05/42.htm

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