Bacteria transformation lab!
During this Lab, we attempted to get foreign genes into an organism. The objective of this lab was to understand that the genetic composition of cells can be altered by incorporation of exogenous DNA into cells to form novel (new) combinations forming GMO genetically modified organisms that are also transgenic.
Question: Can cells be altered to new combinations, forming GMO's that are also transgenic?
Hypothesis: If bacteria cells take in recombinant DNA plasmid, then these cells will be transformed.
Procedure:
1. Label two Eppendorfs- one + and one -
2. Label Plates A, B, C and mark a line down the middle of plates A and B ONLY! On one half label a + and the other half a -
3. On plate C, mark a + on the plate
4. Using a micro pipette set at 050: Add 50ml of CELLS solution to both the + and - tubes
5. Using a second (smaller volume) micro pipette set at 12.0, use a sterile pipette tips, add 12ml of rpARA plasmid to the positive (+) tube only. Tap gently to mix.
6. Incubate both + and - tubes on ice for 10 minutes. This is done to make the cells competent.
7. Take your tubes in the ice water cup to the 42 degrees Celsius hot water bath. Make sure the water bat is 42 degrees Celsius. Transfer the tubes from ice to the hot water for 45-60 seconds HEAT SHOCK. Immediately return the tubes to the ice for 2 minutes.
8. Place + and - tubes in a beaker/cup at room temperature. To normalize bacteria cells- add 150ml LB broth to both tubes. 9. Mix gently and rest tubes for 2 minutes.
10. Set micropipette at 050. Use sterile tip for - loading. Use a new sterile tip for + loading. For the - tube: Add 50ml onto plates A and B. Be sure to place on - side. For the + tube: Add 50ml onto plates A and B. Be sure to place on + side AND 50ml onto plate C.
11.Using a sterile inoculation loop, gently spread the - sides of plates A and B
Question: Can cells be altered to new combinations, forming GMO's that are also transgenic?
Hypothesis: If bacteria cells take in recombinant DNA plasmid, then these cells will be transformed.
Procedure:
1. Label two Eppendorfs- one + and one -
2. Label Plates A, B, C and mark a line down the middle of plates A and B ONLY! On one half label a + and the other half a -
3. On plate C, mark a + on the plate
4. Using a micro pipette set at 050: Add 50ml of CELLS solution to both the + and - tubes
5. Using a second (smaller volume) micro pipette set at 12.0, use a sterile pipette tips, add 12ml of rpARA plasmid to the positive (+) tube only. Tap gently to mix.
6. Incubate both + and - tubes on ice for 10 minutes. This is done to make the cells competent.
7. Take your tubes in the ice water cup to the 42 degrees Celsius hot water bath. Make sure the water bat is 42 degrees Celsius. Transfer the tubes from ice to the hot water for 45-60 seconds HEAT SHOCK. Immediately return the tubes to the ice for 2 minutes.
8. Place + and - tubes in a beaker/cup at room temperature. To normalize bacteria cells- add 150ml LB broth to both tubes. 9. Mix gently and rest tubes for 2 minutes.
10. Set micropipette at 050. Use sterile tip for - loading. Use a new sterile tip for + loading. For the - tube: Add 50ml onto plates A and B. Be sure to place on - side. For the + tube: Add 50ml onto plates A and B. Be sure to place on + side AND 50ml onto plate C.
11.Using a sterile inoculation loop, gently spread the - sides of plates A and B