This is the protocol we have used to transform our E. coli strains.

  • We dilute the plasmid with 15 μl of H20 in the well and then we store it into a pcr tube.
  • We take 1 μl of the chosen plasmid and we mix it with 100 μl of chemically competent bacteria.
  • We use a heat shock protocol with the following steps:
    • 30 minutes in ice
    • 30 seconds at 42ºC (45 sec is also used)
    • 5 minutes in ice
  • Then we put in 900 μl of LB media and leave it at 37ºC during an hour.
  • After the hour we centrifuge it at 3000 rpm during 10 minutes (our centrifuge has a little radius, else we would do it at 1000 rpm).
  • We take the supernatant away, about 900 μl, and resuspend the pellet with the 100 μl left.
  • The last step is to plate it in a Petri dish and incubate it overnight at 37ºC.

The first transformations were made using a BL21 strain, DH5α has been also used, as well as XL1-blue... (the point is to get some plasmids, right?)

We're still discussing which strain will be the chosen one for the characterisation.