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Team:British Columbia/Notebook/Week 3
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Our group was low on competent cells so making more of them was a priority this week. On June 22, Rafael started the protocol by putting 10mL of an overnight culture in an incubator. The cells were DH5(alpha) cells. The next day, Gurpal took over the protocol. It was his first time making competent cells (and performing his own iGEM laboratory work for the first time) and he knew the group needed them - the pressure was on. Gurpal made and autoclaved 1L of LB broth while filter sterilizing 0.1M CaCl2. Then, he grew the overnight culture, in LB broth, in a 30 degree Celsius incubator until a spectrophotomter reading of Abs600nm = 0.401 was achieved. It took about 1.5 hours to reach this point. Next the mixture was spun down using a centrifuge and the supernatant was removed. Next, the pellet was washed in CaCl2 and spun down again. After the cells were kept on ice for 40 minutes, they were spun down again, the supernatant was removed and then Gurpal stored them at 4 degrees Celsius overnight. The next day, on June 24, Laura and Gurpal started an assembly line and stored over 120 aliquots of competent cells in glycerol solutions. The cells were finally stored in a -80 degrees Celsius freezer for later use. | Our group was low on competent cells so making more of them was a priority this week. On June 22, Rafael started the protocol by putting 10mL of an overnight culture in an incubator. The cells were DH5(alpha) cells. The next day, Gurpal took over the protocol. It was his first time making competent cells (and performing his own iGEM laboratory work for the first time) and he knew the group needed them - the pressure was on. Gurpal made and autoclaved 1L of LB broth while filter sterilizing 0.1M CaCl2. Then, he grew the overnight culture, in LB broth, in a 30 degree Celsius incubator until a spectrophotomter reading of Abs600nm = 0.401 was achieved. It took about 1.5 hours to reach this point. Next the mixture was spun down using a centrifuge and the supernatant was removed. Next, the pellet was washed in CaCl2 and spun down again. After the cells were kept on ice for 40 minutes, they were spun down again, the supernatant was removed and then Gurpal stored them at 4 degrees Celsius overnight. The next day, on June 24, Laura and Gurpal started an assembly line and stored over 120 aliquots of competent cells in glycerol solutions. The cells were finally stored in a -80 degrees Celsius freezer for later use. | ||
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| + | Sam and Gurpal made LB agar plates with ampicillin. First, Gurpal prepared a stock ampicillin solution of 100 mg/mL. He added ethanol to the solution so that the ampicillin would not freeze in the -20 degree freezer - so it could be used immediately during an experiment. Next, Sam made LB agar and autoclaved it. When the LB agar cooled down a bit, we added ampicillin into the LB agar and poured ~16 plates. | ||
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| + | In order to check that the plates were working, Sam and Gurpal transformed ampicillin resistant plasmids into ecoli. They had a positive control, a negative control and their sample. They grew the ecoli overnight in a 37 degree incubator. | ||
Revision as of 22:08, 27 July 2011
Our group was low on competent cells so making more of them was a priority this week. On June 22, Rafael started the protocol by putting 10mL of an overnight culture in an incubator. The cells were DH5(alpha) cells. The next day, Gurpal took over the protocol. It was his first time making competent cells (and performing his own iGEM laboratory work for the first time) and he knew the group needed them - the pressure was on. Gurpal made and autoclaved 1L of LB broth while filter sterilizing 0.1M CaCl2. Then, he grew the overnight culture, in LB broth, in a 30 degree Celsius incubator until a spectrophotomter reading of Abs600nm = 0.401 was achieved. It took about 1.5 hours to reach this point. Next the mixture was spun down using a centrifuge and the supernatant was removed. Next, the pellet was washed in CaCl2 and spun down again. After the cells were kept on ice for 40 minutes, they were spun down again, the supernatant was removed and then Gurpal stored them at 4 degrees Celsius overnight. The next day, on June 24, Laura and Gurpal started an assembly line and stored over 120 aliquots of competent cells in glycerol solutions. The cells were finally stored in a -80 degrees Celsius freezer for later use.
Sam and Gurpal made LB agar plates with ampicillin. First, Gurpal prepared a stock ampicillin solution of 100 mg/mL. He added ethanol to the solution so that the ampicillin would not freeze in the -20 degree freezer - so it could be used immediately during an experiment. Next, Sam made LB agar and autoclaved it. When the LB agar cooled down a bit, we added ampicillin into the LB agar and poured ~16 plates.
In order to check that the plates were working, Sam and Gurpal transformed ampicillin resistant plasmids into ecoli. They had a positive control, a negative control and their sample. They grew the ecoli overnight in a 37 degree incubator.
