09/19/14 - Jonah Thomas
- Failed to remove plates containing transformed bacterial cells from incubator and thus, the cells became overgrown
- Measured the concentrations of the available plasmids
- Performed a 1:4 passage of the dox- and dox+ cells in a T-75 flask
Plasmid Concentration Measurement
Note: Measurements taken using Biotek machine - Take 3 plate
- Thawed 6 vials of plasmid at room temperature
- Labeled each vial 1-6, respectively
- Placed 2μL of plasmid on the Take 3 Plate according to Table 1 below:
| Take 3 Plate Well/Vial Number |
Plasmid |
OD260 |
260/280 |
Concentration [ng/μL]
|
| A1 |
Blank |
N/A |
N/A |
N/A
|
| B1/1 |
KAH60/vnPc |
0.07 |
1.82 |
70.44
|
| C1/2 |
KAH60/vnPc |
0.22 |
1.75 |
219.73
|
| D1/3 |
KAH59/MV5 |
0.24 |
1.85 |
241.06
|
| E1/4 |
KAH54/vnPc |
0.36 |
1.79 |
362.24
|
| F1/5 |
KAH54/vnPc |
0.26 |
1.81 |
257.63
|
| G1/6 |
KAH59/vnPc |
0.21 |
1.78 |
213.99
|
Note: It was calculated that 9.102μL of the KAH60/vnPc plasmid (Vial 2) will be needed to reach the 2000ng mass necessary for plasmid for cell transfection. Additionally, there appeared to be ~45-50μL of this plasmid available
Cell Passaging
- HEK293 Gal4-EED cells (-dox and +dox) were passaged into a sterile T-75 flask
- The standard cell passaging protocol was followed
- A 1:4 passage was performed in 1% pen-strep medium
Plan of Action: Week of 9/22/14
- Monday (9/22): Transfer -dox and +dox cells to 6-well plates
- Tuesday (9/23): Check cell growth
- Wednesday (9/24): Perform cell transfections with KAH60/vnPc (Vial 2)
- Be sure to all do mock transfections!
- Depending on cell growth, this may be able to be completed on Tuesday
- Thursday (9/25): Check cell growth
- Friday (9/26): Check cell growth
- On either Thurs. or Friday, the fluorescent microscope will be used to confirm a successful transfection of cells
Note: Upon confirming a successful transfection, a cell count will be completed as well as a luciferase assay
| 
Haynes Lab - Short Projects
