(Source: openwetware ) Author: James Hadfield, CRUK Cambridge Research Institute, Robinson Way, Cambridge CB2 0RE.
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 100μg/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.
Materials
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
Procedure
10μL Ligation Mix
Larger ligation mixes are also commonly used
1.0 μL 10X T4 ligase buffer
6:1 molar ratio of insert to vector (~10ng vector)
Add (8.5 - vector and insert volume)μl ddH2O
0.5 μL T4 Ligase
Calculating Insert Amount
Insert Mass in ng=6 x (insert length in bp / vector length in bp) x 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.