Team:TU Darmstadt/Protocols/DNA Ligation

DNA Ligation

About

DNA ligation is necessary to assemble digested DNA parts into a vector. The cut ends generated by restriciton enzymes are put together by DNA ligase.

Procedure

Ligation (20 µL batch)

• NEB T4 Ligase Buffer 2 µL
• NEB T4 Ligase 0.5 µL
• digested vector 25 ng
• Insert 1:5 molar ratio compared to vector

<poem> Calculation: Insert size (bp) / vector size (bp)= proportion Proportion*vector amount*5= amount of insert for 1:5 ratio </poem>

• Take 10 µL and incubate for 15 minutes at 37°C.
• Take 10 µL and incubate at 4°C over night in order to have a backup ligation batch.

Promega

We recommend using a 1:1, 1:3 or 3:1 molar ratio of vector:insert DNA when cloning a fragment into a plasmid vector. These ratios will vary with other types of vectors, for example, cDNA and genomic cloning vectors. The following example illustrates the conversion of molar ratios to mass ratios for a 3.0kb plasmid and a 0.5kb insert DNA fragment.

ng of vector × kb size of insert × molar ratio of insert = ng of insert kb size of vector vector Example:

How much 0.5kb insert DNA should be added to a ligation in which 100ng of 3kb vector will be used? The desired vector:insert ratio will be 1:3. 100ng vector × 0.5kb insert × 3 = 50ng insert 3kb vector 1 The following ligation reaction of a 3kb vector and a 0.5kb insert DNA uses a 1:1 vector:insert ratio. Typical ligation reactions use 100–200ng of vector DNA.

1. Assemble the following reaction in a sterile microcentrifuge tube:

vector DNA 100ng

insert DNA 17ng

Ligase 10X Buffer 1µl

T4 DNA Ligase (Weiss units) 0.1–1u

Nuclease-Free Water to final volume of 10µl

2. Incubate the reaction at:

room temperature for 3 hours, or

4°C overnight, or

15°C for 4–18 hours.

Notes:

There is considerable latitude in the temperature and time needed for successful ligations. The optimal temperature for a ligation is a balance between the optimal temperature for T4 DNA Ligase enzyme activity (25°C) (1) and the temperature necessary to ensure annealing of the fragment ends, which can vary with the length and base composition of the overhangs. Shorter duplexes (linkers less than 16 bases long) require lower temperatures as a result of their lower melting temperatures (Tm). In general, ligation reactions performed at lower temperatures require longer incubation times. The scientific literature reflects this variability in ligation conditions.

Blunt-end ligations generally are efficient at temperatures between 15–20°C for 4–18 hours, while sticky ends are ligated effectively at room temperature (22°C) for 3 hours or 4–8°C overnight.