Haynes Lab:Notebook/Short Projects/2014/09/19: Difference between revisions

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==09/19/14 - Jonah Thomas==
==09/19/14 - Jonah Thomas==
* Failed to remove plates containing transformed bacterial cells from incubator and thus, the cells became overgrown
* Failed to remove plates containing transformed bacterial cells from incubator and thus, the cells became overgrown
* Obtained 6 vials of plasmid DNA
* Measured the concentrations of the available plasmids
* Measured the concentrations of the available plasmid DNA
* Performed a 1:4 passage of the dox- and dox+ cells in a T-75 flask
* Performed a 1:4 passage of the dox- and dox+ cells in a T-75 flask


----
----


'''Plasmid Concentration Measurement'''
'''Plasmid Concentration Measurement'''
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Note: Measurements taken using Biotek machine - Take 3 plate
Note: Measurements taken using Biotek machine - Take 3 plate


* Thawed 6 vials of plasmid DNA at room temperature
* Thawed 6 vials of plasmid at room temperature
** Labeled each vial 1-6, respectively
** Labeled each vial 1-6, respectively
* Placed 2μL of plasmid on the Take 3 Plate according to Table 1 below:
* Placed 2μL of plasmid on the Take 3 Plate according to Table 1 below:
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{| {{table}}
{| {{table}}
|-
|-
| Take 3 Plate Well || Plasmid || OD260 || 260/280 || Concentration [ng/μL]
| Take 3 Plate Well/Vial Number || Plasmid || OD260 || 260/280 || Concentration [ng/μL]
|-
|-
| A1 || Blank || N/A || N/A || N/A
| A1 || Blank || N/A || N/A || N/A
|-
|-
| B1 || KAH60/vnPc || 0.07 || 1.82 || 70.44
| B1/1 || KAH60/vnPc || 0.07 || 1.82 || 70.44
|-
|-
| C1 || KAH60/vnPc || 0.22 || 1.75 || 219.73
| '''C1/2''' || '''KAH60/vnPc''' || '''0.22''' || '''1.75''' || '''219.73'''
|-
|-
| D1 || KAH59/MV5 || 0.24 || 1.85 || 241.06
| D1/3 || KAH59/MV5 || 0.24 || 1.85 || 241.06
|-
|-
| E1 || KAH54/vnPc || 0.36 || 1.79 || 362.24
| E1/4 || KAH54/vnPc || 0.36 || 1.79 || 362.24
|-
|-
| F1 || KAH54/vnPc || 0.26 || 1.81 || 257.63
| F1/5 || KAH54/vnPc || 0.26 || 1.81 || 257.63
|-
|-
| G1 || KAH59/vnPc || 0.21 || 1.78 || 213.99
| 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'''
* <u>Monday (9/22)</u>: Transfer -dox and +dox cells to 6-well plates
* <u>Tuesday (9/23)</u>: Check cell growth
* <u>Wednesday (9/24)</u>: 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
* <u>Thursday (9/25)</u>: Check cell growth
* <u>Friday (9/26)</u>: 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


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Revision as of 15:34, 19 September 2014

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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



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