Team:EPF-Lausanne/Notebook/17 July 2012

From 2012.igem.org

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(→CHO cell transfection with LOVTAP and RO)
(→CHO cell transfection with LOVTAP and RO)
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{{:Team:EPF-Lausanne/Template/LabPresence|Sowmya, Alexandra, Mouna, June}}
{{:Team:EPF-Lausanne/Template/LabPresence|Sowmya, Alexandra, Mouna, June}}
-
Table
+
Transfection Protocol -> CHO-DG44
 +
 +
*Cell Density 3x10^6cells/mL
 +
*Total Volume 5 mL
 +
*DNA 1.5 µg/10^6cells
 +
*PEI 3 µg/10^6cells
 +
 +
 +
#Passage seed 1 day prior to transfection
 +
#Prepare tubes by addition of DNA
 +
#Centrifuge the necessary volume of seed, remove conditioned medium and resuspend in necessary volume of fresh medium to acheive the required cell density
 +
 +
#Add 5 mL of the cell suspension to the tube with DNA and mix by orbitally shaking
 +
#Add the PEI to the Cell+DNA mixture as soon as possible
 +
#Place in the incubator at 37°C
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{{:Team:EPF-Lausanne/Template/Protocol|Transfection}}
+
 
 +
 
 +
'''Transfection plan:''' different DNA percentages. Each one of those will have a duplicate.
 +
 
 +
'''DNA concentration (Stock values) :'''
 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''LovTAP'''
 +
|'''RO'''
 +
|'''filler'''
 +
|'''unit'''
 +
|-
 +
|0.4
 +
|0.29
 +
|1.06
 +
|µg/µL
 +
|-
 +
|}
 +
 
 +
'''Percentage of DNA'''
 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''Tube number'''
 +
|'''LovTAP'''
 +
|'''RO'''
 +
|'''filler'''
 +
|'''unit'''
 +
|-
 +
|1,2
 +
|100
 +
|0
 +
|0
 +
|%
 +
|-
 +
|3,4
 +
|10
 +
|0
 +
|90
 +
|%
 +
|-
 +
|5,6
 +
|0
 +
|0
 +
|100
 +
|%
 +
|-
 +
|7,8
 +
|50
 +
|50
 +
|0
 +
|%
 +
|-
 +
|9,10
 +
|0
 +
|100
 +
|0
 +
|%
 +
|-
 +
|11,12
 +
|0
 +
|50
 +
|50
 +
|%
 +
|-
 +
|}
 +
 
 +
Mass of DNA
 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''Tube number'''
 +
|'''LovTAP'''
 +
|'''RO'''
 +
|'''filler'''
 +
|'''unit'''
 +
|-
 +
|1,2
 +
|22.5
 +
|0
 +
|0
 +
|µg
 +
|-
 +
|3,4
 +
|2.25
 +
|0
 +
|20.25
 +
|µg
 +
|-
 +
|5,6
 +
|0
 +
|0
 +
|22.5
 +
|µg
 +
|-
 +
|7,8
 +
|11.25
 +
|11.25
 +
|0
 +
|µg
 +
|-
 +
|9,10
 +
|0
 +
|22.5
 +
|0
 +
|µg
 +
|-
 +
|11,12
 +
|0
 +
|11.25
 +
|11.25
 +
|µg
 +
|-
 +
|}
 +
 
 +
'''Final volumes added :'''
 +
{| class="wikitable" style="text-align: center; color: black;"
 +
|'''Tube number'''
 +
|'''LovTAP'''
 +
|'''RO'''
 +
|'''filler'''
 +
|'''unit'''
 +
|-
 +
|1,2
 +
|56.25
 +
|0
 +
|0
 +
|µL
 +
|-
 +
|3,4
 +
|5.625
 +
|0
 +
|18.75
 +
|µL
 +
|-
 +
|5,6
 +
|0
 +
|0
 +
|20.8
 +
|µL
 +
|-
 +
|7,8
 +
|28.125
 +
|38.8
 +
|0
 +
|µL
 +
|-
 +
|9,10
 +
|0
 +
|77.59
 +
|0
 +
|µL
 +
|-
 +
|11,12
 +
|0
 +
|38.8
 +
|10.4
 +
|µL
 +
|-
 +
|}
 +
 
 +
 
 +
{{:Team:EPF-Lausanne/Template/Protocol|None}}
; Comments:
; Comments:

Revision as of 23:08, 18 July 2012



CHO cell transfection with LOVTAP and RO

  • CHO cell transfection with LOVTAP and RO
  • CHO cell new culturing (Will be continued on 19th of July)


Transfection Protocol -> CHO-DG44

  • Cell Density 3x10^6cells/mL
  • Total Volume 5 mL
  • DNA 1.5 µg/10^6cells
  • PEI 3 µg/10^6cells


  1. Passage seed 1 day prior to transfection
  2. Prepare tubes by addition of DNA
  3. Centrifuge the necessary volume of seed, remove conditioned medium and resuspend in necessary volume of fresh medium to acheive the required cell density
  1. Add 5 mL of the cell suspension to the tube with DNA and mix by orbitally shaking
  2. Add the PEI to the Cell+DNA mixture as soon as possible
  3. Place in the incubator at 37°C


Transfection plan: different DNA percentages. Each one of those will have a duplicate.

DNA concentration (Stock values) :

LovTAP RO filler unit
0.4 0.29 1.06 µg/µL

Percentage of DNA

Tube number LovTAP RO filler unit
1,2 100 0 0 %
3,4 10 0 90 %
5,6 0 0 100 %
7,8 50 50 0 %
9,10 0 100 0 %
11,12 0 50 50 %

Mass of DNA

Tube number LovTAP RO filler unit
1,2 22.5 0 0 µg
3,4 2.25 0 20.25 µg
5,6 0 0 22.5 µg
7,8 11.25 11.25 0 µg
9,10 0 22.5 0 µg
11,12 0 11.25 11.25 µg

Final volumes added :

Tube number LovTAP RO filler unit
1,2 56.25 0 0 µL
3,4 5.625 0 18.75 µL
5,6 0 0 20.8 µL
7,8 28.125 38.8 0 µL
9,10 0 77.59 0 µL
11,12 0 38.8 10.4 µL


Protocol: None

Forgot to insert protocol.


Comments

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