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User:Andriana/13 June 2012
From 2012.igem.org
I. 24 Hour Assay Continued
- Centrifuged out pellets of all 4 cell samples grown overnight (all reached the stationary phase due to significant murkiness): in 1.5 mL eppendorf tubes, corresponding cell solutions were added 1 mL at a time, centrifuged, and the supernatant discarded until all cell-broth solutions were used.
- The pellets were washed with 1 mL sterile deionized water 4 times. For the first three washes, a vortex (overtaxing) was used to help resuspend the pellets, but the eppendorf tube was inverted for the 4th wash.
- The fifth time sterile deionized water was added, the pellets were resuspended again with the help of vortex. This solution was used to measure the OD and approximate cell number as follows:
| Cell Type | OD | Cell # approx. (cells/mL) | Volume in microliters for 1 mL of A-F in microliters |
|---|---|---|---|
| 1. NB – normal BL21’s | 0.242 | 2.42x108 | 41.3 |
| 2. NO – normal Origami | 0.265 | 2.65x108 | 37.7 |
| 3. TB – transformed BL21’s | 0.311 | 3.11x108 | 32.2 |
| 4. TO – transformed origami | 0.390 | 3.90x108 | 25.6 |
- (Red Experiment) Estimated 1.0x107 of BL21 cells were added to .2 mL of the following solutions in eppendorf tubes:
- Sterile, deionized water
- .1 M NaCl (20 microliters 5M Nalco + 980 microliters sterile DIW)
- 1 M NaCl (200 microliters 5M NaCl + 800 microliters sterile DIW)
- 5 M NaCl
- .1 M KOH (20 microliters 5 M KOH + 980 microliters DIW)
- .1M CaCl2
II. Shadowed grad students in Isaacs Lab
