Valencia/Transformation
From 2007.igem.org
< Valencia
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.