Sep 23, 2024
T4 Ligation
- 1Washington University
Protocol Citation: Carolina Lopez 2024. T4 Ligation. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71po8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 28, 2024
Last Modified: September 23, 2024
Protocol Integer ID: 100755
Abstract
Protocol for DNA ligation using T4 ligase
Materials
Reagents:
- T4 ligase (Thermofisher, 15224041)
- 5x T4 ligation Buffer
- Competent E. coli cells (NEB, C3040H)
- LB plates with necessary antibiotic
T4 Ligation Protocol (Thermofisher, 15224041):
T4 Ligation Protocol (Thermofisher, 15224041):
1h 39m
1h 39m
T4 DNA Ligase catalyzes the joining of two cohesive- or blunt-ended strands of DNA between the 5´-phosphate and the 3´-hydroxyl groups of adjacent nucleotides.
1. For initial reaction, Mix:
X and Y should be calculated for 3 Insert:1 vector molar ratio
Example of Molar Ratio calculation for 3000bp vector with 500bp PCR product insert:
| A | B | C | D | E | |
| Component | Length of DNA (bp) | Molar ratio | ng of DNA | Volume of 50ng/ul solution | |
| Vector | 3000 | 1 | 50 | 1 ul | |
| PCR Fragment | 500 | 3 | 25 | 0.5 ul | |
| H20 | 8.5 ul |
2. Incubate at room temperature for 00:15:00 .
3. Transform Product into E. coli
a) Add 2 µL of product to 50 µL of TOP10 cells and pipette up and down slowly to mix.
b) Incubate for 00:20:00 on ice.
c) Heat shock bacteria in 42 °C waterbath for 00:01:00 .
d) Incubate on Ice for 00:03:00 .
e) Add 100 µL of SOC media and shake in warm room for 01:00:00 .
f) Plate bacteria onto LB-Antibiotic Plate and spread cells with plate spreader to get individual colonies.
g) Incubate overnight in 37 °C warm room.
h) Pick colonies for miniprep growth and sequencing.
1h 39m