Transforming into DH5α or TOP10 E. coli

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).




 * 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 )