Transforming into DH5α or TOP10 E. coli

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When transforming into DH5-alpha E.coli cells we followed the protocol below:

The plasmids we transformed were present in a 25µl ligation mixture (6µl of this being the vector plasmid, and 15µl of this being the digested plasmid in which the desired 2.2kb fragment was restricted out of).

Preparing our transformations on ice


  • On ice, add 12.5µl of the ligation mixture (see Ligating DNA protocol) to 100µl of DH5-alpha E.coli cells in an eppendorf tube
  • Mix well by pipetting up and down several times
  • Leave on ice for 45 minutes
  • Remove from ice and incubate for 3 minutes at 37˚C (this is the heat shock)
  • Add 300µl of LB and mix well with pipette
  • Incubate for 1 hour in a 37˚C water bath
  • Plate onto antibiotic agar (see Making Agar Plates )


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