Oct 06, 2022
Version 2
Plasmid transduction using competent cell V.2
- 1XJTLU
Protocol Citation: An.Huang 2022. Plasmid transduction using competent cell. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw4e5l5r/v2Version created by An.Huang
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: October 06, 2022
Last Modified: October 06, 2022
Protocol Integer ID: 70907
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Abstract
Plasmid can be transduced into bacteria at competent state using heat shock. This protocol helps transduce plasmid into competent cells.
Materials
Competent cell, DNA plasmid solution, LB broth medium, LB agar plate (with antibiotics)
Take competent cell out from -80℃ fridge and thaw on ice.
When the cells are completely thawed, pipette 2 µL plasmid DNA solution into 100 µL competent cell.
Put the cell in ice for 00:30:00
30m
Conduct heat shock on the competent cell by placing the cell in 42 °C water bath for 00:01:30 .
Put the cells back into ice for 00:02:00
3m 30s
Add 900 µL LB broth medium into competent cell mixture. Shake at 180 rpm, 37°C for 00:45:00
45m
Centrifuge at 6000 rpm, Room temperature, 00:05:00。.
Note
Centrifuge radius = 6 cm.
5m
Discard 900 µL supernatant and resuspend the pellet in the rest 100 µL supernatant.
Spread the cells onto LB agar plates.
Note
LB agar plates may contain antibiotics, which is determined by the transduced plasmid.
Place the plate with lid on upside for 01:00:00 .
1h
Invert the plate and culture at 37 °C in a biomedical incubator overnight.
Note
If the bacteria turn out to be too concentrated, dilute the cell before spreading on the plate next time.