Trial 2 Liquid Culture 4 of BL21DE Bacteria Cells
3-25-14
Started Culture at 37 C at 7:34 am.
Took 3 1mL samples from our 50mL of non-induced bacteria. We put it through a spectrophotometer to see how much murky the sample. The more murky it is, the more bacteria colonies there are. After the sample was close to .600 we removed 7mL of non-induced sample and made that our control. Now we have 40mL of our original non-induced culture. We added .4mL of IPTG, which is the sugar that turns on our gene. Afterwards, we took that culture and shook it in the 37 C shaker. Then we placed it in 4 C.
3-26-14
We took 40mL of the induced culture and put it in 4 15mL conical tubes. Then we made a dummy tube of 7mL of water to balance our control tube and put all 6 tubes in the centrifuge for 20 minutes at 4000 rpm in 4 C. After it was finished, we extracted the supernatant from all of the tubes. Then we put the TRIS buffer to the pellets of the cultures and re-suspend the cells. Once again, we took the 4 induced bacteria tubes, the control, and the dummy tubes for 20 minutes at 4000 rpm in 4 C. When it when finished, we got rid of the supernatant, saved the pellet, labeled our tubes and placed it in -20 C. (We were supposed to put pellets in -80 C, but we didn't have those conditions.
3-25-14
Started Culture at 37 C at 7:34 am.
Took 3 1mL samples from our 50mL of non-induced bacteria. We put it through a spectrophotometer to see how much murky the sample. The more murky it is, the more bacteria colonies there are. After the sample was close to .600 we removed 7mL of non-induced sample and made that our control. Now we have 40mL of our original non-induced culture. We added .4mL of IPTG, which is the sugar that turns on our gene. Afterwards, we took that culture and shook it in the 37 C shaker. Then we placed it in 4 C.
3-26-14
We took 40mL of the induced culture and put it in 4 15mL conical tubes. Then we made a dummy tube of 7mL of water to balance our control tube and put all 6 tubes in the centrifuge for 20 minutes at 4000 rpm in 4 C. After it was finished, we extracted the supernatant from all of the tubes. Then we put the TRIS buffer to the pellets of the cultures and re-suspend the cells. Once again, we took the 4 induced bacteria tubes, the control, and the dummy tubes for 20 minutes at 4000 rpm in 4 C. When it when finished, we got rid of the supernatant, saved the pellet, labeled our tubes and placed it in -20 C. (We were supposed to put pellets in -80 C, but we didn't have those conditions.