User:Jarle Pahr/Ligation
Notes on DNA ligation:
http://bitesizebio.com/articles/the-basics-how-does-dna-ligation-work/
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 |
| Addgene | 25 ng | 3:1 | RT 2 h or 4C ON | http://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.
