DNA ligation

Curators
James Hadfield, CRUK Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE.

''Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. This is a new initiative on OWW, please provide your thoughts on the idea of consensus protocol curators here.''

Abstract
This is a consensus protocol. See the bottom of this article for specific protocols.

DNA ligation is the process of joining together two DNA molecule ends (either from the same or different molecules). Specifically, it involves creating a phosphodiester bond bond between the 3' hydroxyl of one nucleotide and the 5' phosphate of another. This reaction is usually catalyzed by a DNA ligase enzyme. This enzyme will ligate DNA fragments having blunt or overhanging, complementary, 'sticky' ends. Typically, it is easier to ligate molecules with complementary sticky ends than blunt ends. T4 DNA ligase is the most commonly used DNA ligase for molecular biology techniques and can ligate 'sticky' or blunt ends.

The two components of the DNA in the ligation reaction should be equimolar and around 100&mu;g/ml. Most commonly, one wants to ligate an insert DNA molecule into a plasmid, ready for bacterial transformation. Typically, DNA and plasmid vector are individually cut to yield complementary ends, then both are added to a ligation reaction to be circularised by DNA ligase. If the plasmid backbone to insert DNA ratio is too high then excess 'empty' mono and polymeric plasmids will be generated. If the ratio is too low then the result may be an excess of linear and circular homo- and heteropolymers.

Reagents

 * T4 DNA ligase
 * 10x T4 DNA Ligase Buffer
 * Deionized, sterile H2O
 * Purified, linearized vector (likely in H2O or EB)
 * Purified, linearized insert (likely in H2O or EB)

Equipment
Vortex

10&mu;L Ligation Mix
Larger ligation mixes are also commonly used
 * 1.0 &mu;L 10X T4 ligase buffer
 * 6:1 molar ratio of insert to vector (~10ng vector)
 * Add (8.5 - vector and insert volume)&mu;l ddH2O
 * 0.5 &mu;L T4 Ligase

Calculating Insert Amount
$${\rm Insert\ Mass\ in\ ng} = 6\times\left[\frac\right]\times{\rm Vector\ Mass\ in\ ng} $$

'''The insert to vector molar ratio can have a significant effect on the outcome of a ligation and subsequent transformation step. Molar ratios can vary from a 1:1 insert to vector molar ratio to 10:1. It may be necessary to try several ratios in parallel for best results.'''

Method

 * 1) Add appropriate amount of deionized H2O to sterile 0.6 mL tube
 * 2) Add 1 &mu;L ligation buffer to the tube. Vortex buffer before pipetting to ensure that it is well-mixed. Remember that the buffer contains ATP so repeated freeze, thaw cycles can degrade the ATP thereby decreasing the efficiency of ligation.
 * 3) Add appropriate amount of insert to the tube.
 * 4) Add appropriate amount of vector to the tube.
 * 5) Add 0.5 &mu;L ligase. Vortex ligase before pipetting to ensure that it is well-mixed. Also, the ligase, like most enzymes, is in some percentage of glycerol which tends to stick to the sides of your tip.  To ensure you add only 0.5 &mu;L, just touch your tip to the surface of the liquid when pipetting.
 * 6) Let the 10 &mu;L solution sit at 22.5&deg;C for 30 mins
 * 7) Denature the ligase at 65&deg;C for 10min
 * 8) Dialyze for 20 minutes if electroporating
 * 9) Use disks shiny side up
 * 10) Store at -20&deg;C

Factors affecting efficiency
From Tom Ellis

A protocol analysis experiment for a typical DNA ligation (7.2 kb vector + 0.6 kb insert, sticky ends) gave optimal ligation efficiency when 50 ng of vector was ligated overnight at 16&deg;C with a 2:1 insert:vector molar ratio and standard T4 ligase. Ligase was heat inactivated at 65&deg;C for 20 mins before 2 &mu;L (of 20 &mu;L) was used to transform commercial heat-shock competent cells.

Ligation efficiency was marginally decreased by
 * 1) Doing a 1 hr ligation at room temperature
 * 2) Using 100 ng vector
 * 3) Using insert:vector molar ratios of 5:1 and 1:1

Ligation efficiency was noticably decreased (x100) by
 * 1) Sticky end ligation with a larger insert (5.2 kb vector + 2.6 kb insert)
 * 2) Blunt end ligation

Ligation efficiency was severely decreased (x10000) by
 * 1) Using DNA fragments that have been exposed to UV during the gel extraction procedure (can avoid by blind excision, or by using a black-light or 365nm UV transilluminator instead of the usual 312nm type)
 * 2) Using the NEB Quick Ligation Kit (heat inactivation of PEG in the buffer ruins transformation, without heat inactivation the ligation probably would've been fine)

For additional troublshooting, check out the NEB FAQ page for T4 ligation:

Acknowledgments
This protocol is primarily based on Endy:DNA ligation using T4 DNA ligase.

Specific protocols
Endy:DNA ligation using T4 DNA ligase -- Using T4 DNA Ligase

Knight:DNA ligation using NEB Quick Ligation Kit -- 5min ligation.

Knight:TOPO TA cloning -- For PCR products.

Silver:Ligation -- A protocol for sticky end ligations using the Roche Kit.

Richard_Lab:Ligation -- Uses T4 Ligase

BE.109:DNA ligation -- A ligation protocol for classroom use in a laboratory class taught at MIT. Uses T4 DNA ligase but has interesting tips and tricks.

Discussion
You can discuss this protocol.