Ligation with T4 DNA Ligase
Component |
20 uL rxn |
10X T4 DNA Ligase Buffer |
2 uL |
RNase-free Water |
Fill to 20 uL |
Vector DNA |
50 ng* |
Insert DNA |
variable* |
T4 DNA Ligase |
1 uL |
Incubate for 10 min at room temperature before continuing to transformation.
*Using NEBiocalculator, calculate the required mass of insert DNA using 50 ng of vector DNA. Use the mass required for a 1:3 vector to insert ratio. DNA concentrations can vary, but NEB does not recommend overall DNA concentrations exceeding 10 ng/uL (200 ng for a 20 uL reaction).
Heat Shock Transformation of NEB 5-alpha E. coli
- Inoculate 3 mL of LB media in a culture tube with NEB 5-alpha cells and grow overnight at 37 C with shaking.
- Add 30 uL of the overnight culture to 3 mL of fresh LB in a new culture tube 1.5 - 2 hours before you start the transformation. Incubate with shaking.
- Turn on water bath to start warming up to 42C
- Grow cells until the culture reaches an OD600 of 0.3-0.4. This should take around 1-1.5 hours of growth.
- Remove the cells from the incubator. Transfer the culture to a 1.5 mL microcentrifuge tube and pellet the cells by centrifuging at 4,000 rcf for 1 min. Repeat until all cells are pelleted.
- Resuspend the pellet in 1 mL of chilled CaCl2.
- Centrifuge for 1 min at 4,000 rcf
- Dump out supernatant liquid, leaving the pellet at the bottom.
- Resuspend the pellet in 50 uL of CaCl2 per transformation you are going to do. If you are going to do four transformations, for instance, add 200 uL of CaCl2.
- Pipette 50 uL of competent cells into a microcentrifuge tube for each transformation.
- Pipette 10 uL of ligation reaction into the corresponding microcentrifuge tube.
- Sit tubes on ice 30 minutes
- Heat shock tubes in water bath at 42 C for 45 seconds
- Immediately transfer tubes back on ice and let them sit for two minutes
- Pipette 500 uL of LB media to each tube
- Incubate for 1 hour at 37 C with shaking.
- Plate the transformations on LB agar plates with the appropriate antibiotic and incubate overnight at 37 C without shaking.