Haynes Lab:Notebook/Short Projects/2014/09/19

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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