Home›Microbiology›How to Perform a Transformation with NEB Competent Cells
Steps
1Thaw competent cells and prepare DNA00:12
2Mix DNA with competent cells00:21
3Incubate mixture on ice00:30
4Perform heat shock treatment00:37
5Cool cells on ice and add recovery media00:44
6Incubate transformation mixture with shaking01:04
7Plate transformation mix on selection media01:15
8Incubate plates overnight for colony growth01:32
MicrobiologyNew England Biolabs
How to Perform a Transformation with NEB Competent Cells
Protocol
Difficulty
intermediate
Steps
1
Thaw competent cells and prepare DNA
Thaw one tube of NEB competent cells on ice and place your DNA on ice to keep both components cold before transformation.
▶ 00:12
2
Mix DNA with competent cells
Add DNA to 50 µl of competent cells and gently mix by pipetting up and down or flicking the tube 4-5 times. Do not vortex.
▶ 00:21
3
Incubate mixture on ice
Incubate the DNA-competent cell mixture on ice for 30 minutes without mixing to allow DNA uptake.
▶ 00:30
4
Perform heat shock treatment
Heat shock the mixture at 42°C for 10-30 seconds depending on strain without mixing to induce DNA uptake into cells.
▶ 00:37
5
Cool cells on ice and add recovery media
Incubate the mixture on ice for 5 minutes, then add 950 µl of room-temperature SOC media to allow cells to recover.
▶ 00:44
6
Incubate transformation mixture with shaking
Incubate the transformation mixture at 37°C for 60 minutes while shaking vigorously at 250 rpm or rotating to allow cells to express antibiotic resistance.
▶ 01:04
7
Plate transformation mix on selection media
Spread 50-100 µl of the transformation mixture onto warmed selection plates, optionally plating multiple concentrations to ensure optimal colony counts.
▶ 01:15
8
Incubate plates overnight for colony growth
Incubate the plates overnight at 37°C to allow transformed colonies to grow and appear on the selection plates by morning.