Tuesday 4th May 2010
I performed two transformations (with negative and positive controls) by heat shock with competent cells from Miguel’s lab.
- TetR repressible promoter (BBa_R0040), witch is in the Kit plate 1, well 6I from the 2009 distribution. It is on plasmid pSB1A2 resistant to ampicillin.
- Pops-> TetR[S0151], from Kit Plate 1, well 10I. It is on plasmid pSB1A2 resistant to ampicillin.
- Positive control: Luciferase (Mariana) with resistant to kanamycin but not to ampicillin.
- Negative control: No DNA just the competent cells.
When extracting the DNA from the 2009 distribution I was a little confused and I added 40 ul of water to well 10I and 20 for well 6I, that’s why when transforming I added 10 instead of 5 ul for the 10I tranformation.
Heat shock transformation:
- Take 50ul of competent cells from Miguel’s lab frozen at -70ºC, out of a 100ul eppendorf into a new one (sterilized), then I add 5ul of the DNA and put into ice for 20 minutes.
- After that give it a heat shock at 42 ºC for one minute, then put it in ice for another 5 minutes.
- Now grow it in 1ml of liquid LB medium with no antibiotic and shaking for one hour.
- Then centrifuge for 1 minute at 13 rpm and keep the pellet with 100ul, now plate those 100ul in a petri box with ampicillin using the triangle and put them in the incubator at 37ºC overnight.
Note: when extracting DNA from the iGem kit plates add 15ul of water, NO more!!!