Notes on DNA ligation:
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.
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
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/
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/|
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.
~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.
Phosphateses such as Calf Intestinal Phosphatase can be used to dephosporylate DNA.