The Procedure

Tithi Paul

1. Two micro test tubes were obtained, one was labelled +pGLO and the other was labelled –pGLO.
2. Using sterile transfer pipet, 250 µl of transformation solution (CaCl2) was added to each test tube and the transfer pipet was placed in the beaker of bleach solution.
3. The tubes were placed on a foam micro centrifuge tube holder, which was then placed on ice.
4. Using a sterile loop, 2-4 large colonies of bacteria, which are circular with smooth edges, were scraped off the starter plate.
5. The sterile loop was immersed into the solution in the +pGLO test tube and spun between the thumb and index finger a couple times (until the entire colony was dispersed in the transformation solution).
6.  Steps 4 and 5 were repeated for –pGLO test tube using a new sterile loop.
7. Another new sterile loop was immersed into the pGLO plasmid DNA stock tube and a loopful was taken out and mixed into the +pGLO tube.
8. The tubes were returned to the rack on ice and incubated there for 10 minutes.
9. The LB nutrient agar plates were labelled as: LB -, LB/amp +, LB/amp –, and +LB/amp/ara and with the group numbers
10. Both of the test tubes, with the foam rack as a holder, were transferred to a water bath with a temperature of 42∘C for exactly 50 seconds.
11. The test tubes were then incubated on ice for another 2 min.
12. The rack with the test tubes were then placed on the lab bench top and 250 µl of LB nutrient broth was added to each tube with a new sterile pipet.
13. Using a new sterile pipet for each tube, 100 µl of the transformation and control suspensions were added to the appropriate nutrient agar plates.
14. Using a new sterile loop, the suspensions were evenly spread around the surface of the LB nutrient agar by crosshatching.
15. The plates were stacked up (negatives at the bottom and positives on top) and taped together.
16. The stack of plates were flipped upside down and incubated in the incubator at 37∘Celsius for two days (over the weekend).