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- Glida: 1
- WOW!!!
- Glida: 2
- Bacteria transformation is the main method in molecular cloning to produce multiple copies of a specific DNA.
- No! did I just clone myself?
- YE
- Glida: 3
- Step 1:Label the micro test tubes +pGLO and -pGLO respectively, and add 250μl of transformation solution through a sterile transfer pipette into each tube (we're adding CaC1_2).
- EXAMPLE: pGLO
- This lab acted as an visual representation of how this bacterium (pGLO) transform.
- + pGLO
- -pGLO
- 250μl
- Glida: 4
- Step 2: Put the test tubes in separate sections of the crushed ice (cubed ice simply for illustration, please do not use cubed ice my fellow students)
- here
- Glida: 5
- In 1928, in attempt to develop a type of vaccine against pneumonia, Frederick Griffith became the first to discover bacterial transformation, in which the form as well as the function of the bacteria alters to serve a specific purpose (Shen, Griffith's experiment 2023).
- Step 3:Use a sterile loop to collect a single colony of the bacteria from the starter plate, and fully disperse it by immersing the loop at the bottom of both the +pGLO and -pGLO tubes (see the picture above as a visual guide for my smart students).
- Glida: 6
- Step 4: Acquire pGLO plasmid DNA, you can examine it with a UV lamp. After that, immerse a new sterile loop into the solution and carry it exclusively to the +pGLO tube. Or you can use a pipette to extract and add 10μl of the pGLO plasmid solution to the test tube.
- Glida: 7
- Step 5:Put both the +pGLO tube and -pGLO tube on ice for 10 minutes, must fully incubate the test tubes.
- Glida: 8
- Step 6:Label the four agar plates, that's +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp, -pGLO LB respectively. Then after 10 minutes take out the test tubes and give them a heat shock by putting them into a 42 degrees Celsius water bath for 50 seconds, then after 50 seconds put the test tubes back in the ice for another 2 minutes.
- 10 minutes later...
- Glida: 9
- 100μl
- Step 7:Extract the tubes from the ice bath, then use a new sterile pipette to add 250μl of LB nutrient to both +pGLO and -pGLO tubes, then leave the tubes at room temperature without any external influences for 10 minutes. After 10 minutes, use a new sterile pipette to extract 100μl of either +pGLO or -pGLO corresponding to the labelled agar plates.
- Glida: 10
- Step 8:Use new sterile loops for each plate, and equally spread the LB nutrient solution by moving in a back forth action. Then close the lids of each agar plate, and stack them upside down. After all of the steps above, put the plates in the 37 degrees Celcius incubator for at least 24 hours.
- Glida: 11
- Here's my hypothesis for the results, I expect to see relatively less bacterial growth on each plate, but there will be subtle differences between the plates. The bacteria will display a milky white color, also it will produce an odor because it's molding.STUDENTS TAKE NOTES
- Glida: 12
- Here are the actual observations: For +pGLO LB/amp, the bacterium congregates in small areas, producing separated small dots, and creating a stench. For +pGLO LB/amp/ara, the bacterium congregates in separated but relatively close areas, which also creates a stench. For -pGLO LB/amp, no visible bacterium is displayed within the plate producing a subtle odor. Finally, for -pGLO LB, Bacterium fills up the whole plate, and the bacteria stack on top of each other, producing the strongest stench out of all four plates.
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