Ligation Protocol WITH T4 DNA Ligase (M0202)
Updated 4/28/2019 03:37 AM
Introduction
Materials
- Vector DNA (4kb)
- Insert DNA (1kb)
- Nuclease-free water
Procedure
Set up the T4 DNA Ligase Reaction
Note: T4 DNA Ligase should be added last. The table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes. Use NEB calculator to calculate molar ratios.
- Thaw the T4 DNA Ligase Buffer and resuspend at room temperature.
Tip: Alicuote the 10x buffer less concentrated so when thawing, the DTT gets soluble more easily.
- Set up the following reaction in a microcentrifuge tube on ice:
Table1
A A | B B | |
1 1 | Component | Volume (μl) |
2 2 | 10X T4 DNA Ligase Buffer | 2 |
3 3 | Vector DNA: 50 ng (0.020 pmol) | |
4 4 | Insert DNA: 37.5 ng (0.060 pmol) | |
5 5 | Nuclease-free water | 17 |
6 6 | T4 DNA Ligase | 1 |
7 7 | Total | 20 |
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours.
- Heat inactivate at 65 degrees C for 10 minutes.
- Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
Use 25µL DH5α cells, and add 2µL of reaction mixture.
Please see the NEB website for supporting information on this protocol.