User:Jarle Pahr/Ligation

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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.

See also

iGEM protocol:

Protocol, oomocyteworld:


Protocol, NEB T4 DNA ligase:

Protocol,D.W Andrews lab:

Protocol, Biologicalworld:


Protocol, Addgene:


Comparison of protocols:

Protocol source Vector amount Insert:vector ratio Incubation Reference
Biologicalworld 200ng - 14-16 C ON
iGEM 25 ng 1:1 16 C, 30 min
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.
NEB 50 ng (3kb vecotr) 3:1 16 C ON or Room temp 10 min
Addgene 25 ng 3:1 RT 2 h or 4C ON

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.


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