Protocol for Hi-T4™ DNA Ligase (NEB #M2622)

1. Set up the following reaction in a microcentrifuge tube on ice. (Hi-T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.)

Use NEBiocalculator to calculate molar ratios.

Cohesive End Ligation:

COMPONENTS

20 µl REACTION

 

Blunt/TA Overhang Ligation:

COMPONENTS

20 µl REACTION

T4 DNA Ligase

Reaction Buffer (10X)*

2 µl

 

StickTogether™ DNA Ligase Buffer (2X)

10 µl

Vector DNA (4 kb)

50 ng (0.020 pmol)

 

Vector DNA (4 kb)

50 ng (0.020 pmol)

Insert DNA (1 kb)

37.5 ng (0.060 pmol)

 

Insert DNA (1 kb)

37.5 ng (0.060 pmol)

Nuclease-free water

to 20 µl

 

Nuclease-free water

to 20 µl

Hi-T4 DNA Ligase

1 µl

 

Hi-T4 DNA Ligase

1 µl

 

 *The T4 DNA Ligase Reaction Buffer should be thawed and resuspended at room temperature. 

2. Gently mix the reaction by pipetting up and down and microfuge briefly.

3. For cohesive (sticky) ends (1X T4 DNA Ligase Buffer), incubate between 25-50°C for 10 minutes. Heat inactivate at 65°C for 10 minutes.

4. For blunt ends or single base overhangs (1X StickTogether™ DNA Ligase Buffer),  incubate between 25-50°C for 10 minutes or 16°C overnight. Do not heat kill the reaction because heat treating the PEG in the StickTogether™ DNA Ligase Buffer will inhibit transformation.

5. Chill on ice and transform 1-5 µl of the reaction into 50 µl competent cells.