Friday, April 15, 2011

Transformation Lab

Steps to the transformation lab:
1.) The first step was to get organized, and label all of our material. We labeled a green tube with a (+) for +pGLO and on orange tube with a (-) for -pGLO. The tubes were then placed into a foam rack, until needed. 
2.) Having both the green and the orange tube open, we used a sterile transfer pipet to transfer 250 ul of (CaCl2) also known as the transformation solution. After both tubes being filled with the transformation solution, the tubes were placed on ice to prepare for the next step.
3.) We then used a sterile loop to pick up a colony of bacteria from a starter plate. We placed an unknown amount of colonies in both the green (+) and the orange (-) tubes. 
4.) Soon after, we found ourselves waiting for the plasmid DNA solution. With a new sterile loop, it was a difficult task to get the plasma to fill the loop. Once it was finally filled, we inserted the plasma into the green tube (+) but not the orange tube (-). 
5.) The tubes were then placed in ice for 10 minutes. While we waited for the tubes on ice, we labeled our 4 agar plates with: LB/amp (+), LB/amp (-), LB/amp/ara, and LB.
6.) The tubes were then placed into the foam rack and placed into heat shock of 42 degrees for 50 seconds. After the 50 seconds were up, the tubes were placed back into the ice container for 2 minutes. 
7.) Once the 2 minutes were up, we then placed 100 ul of LB nutrient broth in both tubes. The tubes were then incubated for 10 minutes at room temp. 
8.) After the 10 minutes were up, we then used a sterile pipet for each plate, and added 100 ul of transformation and control suspensions into the appropriate plates. We then spread the suspensions evenly over the surface.
9.) The plates were then stacked together, and the stack was placed in the incubator at 37 degrees until the next day.

We expected to find the most bacteria on the LB, because it wasn't tampered with as much. We decided that the LB/amp and the LB/amp/ara would have genetically transformed bacterial cells, due to the fact that they have antibiotics. The +pGLO amp and the -pGLO amp we decided should be compared to determine if there was any genetic transformation that occurred. 

We drew what we saw on each plate, and placed our information into a data table. The -pGLO LB/amp came up with no colonies. The +pGLO LB/amp we found 2 colonies. The +pGLO LB/amp/ara had 6 colonies. The -pGLO LB had 8 colonies.

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