Aug 06, 2024
Competent Transformation
- Miquel Estévez-Gay1
- 1Universitat de Girona
Protocol Citation: Miquel Estévez-Gay 2024. Competent Transformation. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl82w47l2w/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: August 06, 2024
Last Modified: August 06, 2024
Protocol Integer ID: 104804
Keywords: plasmid, competent transformation transform, cell
Abstract
Transform a Plasmid into previously obtained Competent E.coli Cells.
Safety warnings
Competent Cells are fragile. Please do not use Vortex and work with aseptic conditions
Melt in ice and aliquot of 100μL competent E-coli DH5α or BL21(DE3) .
Preheat 500 µL LB or SOC medium (37ºC).
Add 50-100μg DNA into competent cells and mix softly. This is usually 1-2µl from minipreps.
I recommend using 10µl if the DNA comes from PCR and/or DpnI digestion.
Incubate 00:20:00 in ice.
20m
Incubate without shaking at 42 °C for00:01:00 .
1m
Incubate 00:02:00 in ice.
2m
Add 500 µL LB or SOC pre-heated media. This must be done under Laminar Flux Cabin
Incubate 01:00:00 800 rpm, 37°C in shaker thermal block.
1h
Prepare Amp or Kan plates.
Add 100 µL competent cells into each plate and plate. Mix using sterile glass balls under laminar flux cabin.
Incubate 37 °C Overnight
Next day, store the plates parafilmed in the fridge. Can be stored for several weeks.