Competent Transformation
Miquel Estévez-Gay
Abstract
Transform a Plasmid into previously obtained Competent E.coli Cells.
Steps
Melt in ice and aliquot of 100μL competent E-coli DH5α or BL21(DE3) .
Preheat 500µL 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 0h 20m 0s in ice.
Incubate without shaking at 42°C for0h 1m 0s.
Incubate 0h 2m 0s in ice.
Add 500µL LB or SOC pre-heated media. This must be done under Laminar Flux Cabin
Incubate 1h 0m 0s``800rpm in shaker thermal block.
Prepare Amp or Kan plates.
Add 100µLcompetent cells into each plate and plate. Mix using sterile glass balls under laminar flux cabin.
Incubate 37°C``1h 0m 0s
Next day, store the plates parafilmed in the fridge. Can be stored for several weeks.
