Team:iTesla-SoundBio/LigationandTransformationwithNEB5-alphaE.coli

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

    1. Inoculate 3 mL of LB media in a culture tube with NEB 5-alpha cells and grow overnight at 37 C with shaking.
    2. 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

 

  1. Grow cells until the culture reaches an OD600 of 0.3-0.4. This should take around 1-1.5 hours of growth.
  2. 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.
  3. Resuspend the pellet in 1 mL of chilled CaCl2.
  4. Centrifuge for 1 min at 4,000 rcf
  5. Dump out supernatant liquid, leaving the pellet at the bottom.
  6. 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.
  7. Pipette 50 uL of competent cells into a microcentrifuge tube for each transformation.
  8. Pipette 10 uL of ligation reaction into the corresponding microcentrifuge tube.
  9. Sit tubes on ice 30 minutes
  10. Heat shock tubes in water bath at 42 C for 45 seconds
  11. Immediately transfer tubes back on ice and let them sit for two minutes
  12. Pipette 500 uL of LB media to each tube
  13. Incubate for 1 hour at 37 C with shaking.
  14. Plate the transformations on LB agar plates with the appropriate antibiotic and incubate overnight at 37 C without shaking.