Protocol Citation: SGD 2023. Identification of a Plasmid: Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5jmj6l1b/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: February 20, 2023
Last Modified: February 21, 2023
Protocol Integer ID: 77305
Abstract
Protocol for the identification of a plasmid by using transformation of E. Coli
Transformation
Transformation
30s
30s
Transfer 1 mL E. Coli to each of two 1.5 mL Eppendorf tube
Centrifuge at full speed for 00:00:30 , then remove the supernatant
30s
Add 450 µL of TFB1 to each tube, then resuspend gently by pipetting up and down (do not vortex)
Keep everything On ice
Incubate on ice for 00:10:00
10m
Pellet the bacteria by centrifugation at full speed for 00:00:30
Remove the supernatant
Resuspend the pellets in 100 µL of TFB2
Incubate on On ice for 00:10:00
10m 30s
Add 100 ng of plasmid DNA to the competent cells, and mix gently
Heat shock the cells by transferring directly from the ice to the 37 °C hot block for 00:05:00
Then incubate on ice for 00:05:00 , and add 1 mL LB broth to each tube
10m
Incubate again at 37 °C , for 00:30:00 , and allow the cells to recover
30m
Pellet the bacteria again by centrifuging at full speed for 00:00:30
Discard the supernatant
30s
Resuspend the pellet in 400 µL of LB broth
Spread 100 µL of cells per plate, onto the appropriate antibiotic plates
Incubate the plates Overnight at 37 °C
30s