Team:Heidelberg/Notebook/Measurement Standard

From 2010.igem.org

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=Measurement Standard=
=Measurement Standard=
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This page is still under construction.
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'''[[Igem2010/Main|⇐ Main Page]]'''
 +
 
 +
'''[[Igem2010/Main/Measurements/Notebook|⇐ Measurements]]'''
 +
 
 +
----
 +
 
 +
=08/08/2010=
 +
 
 +
seeding cells for test measurements - 96 well plate for plate-reader and FACS
 +
 
 +
*cells were grown in DMEM 10%FBS with phenol red
 +
*washed with PBS
 +
*trypsinised (2 ml trypsin)
 +
*5ml of OptiMEM media (no FBS) added
 +
*counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
 +
*seeded 5000 and 2500 cells/well as on the scheme [[96well plate 080810.jpg]]
 +
*media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
 +
*in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids
 +
 +
-> cells did not adhere well, coat plate with poly-L-lysine and repeat
 +
 
 +
----
 +
 
 +
=09/08/2010=
 +
 
 +
seeding and transfecting cells for microscopy test measurements
 +
 
 +
* add 0.6ul FuGENE reagent to 20ul of OptiMEM
 +
* mix and incubate 5' at RT
 +
* add 0.2ug DNA
 +
* mix and incubate 15' at RT
 +
* add DNA-FuGENE solution to 10 000 cells (400ul)
 +
* mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
 +
* transfere cells to the plate
 +
* grow 48h
 +
 
 +
 
 +
{| class="wikitable" style="border:solid 1px #AAAAAA; border-collapse:collapse;  background-color:#F9F9F9; font-size:95%; empty-cells:show;"
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| style="text-align:right" | HeLa, EGPF || HeLa, EBFP2 || HeLa, EGPF and EBFP2 ||  HeLa, EGPF and EBFP2 1:10
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|-
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| style="text-align:right" | HEK T-REx, EGFP || HEK T-REx, EBFP2 || HEK T-REx, EGPF and EBFP2 || HEK T-REx, EGPF and EBFP2 1:10
 +
|}
 +
 
 +
----
 +
 
 +
=10/08/2010=
 +
 
 +
coating plates with poly-L-lysine
 +
* add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
 +
* leave for 30' in the incubator
 +
* remove poly-L-lysine solution
 +
* wash once with PBS
 +
 
 +
new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously
 +
 
 +
----
 +
 
 +
=25/08/2010=
 +
 
 +
seeding cells for pilot FACS and Tecan measurements (96-well plate)
 +
 
 +
----
 +
 
 +
=26/08/2010=
 +
 
 +
transfection - 96-well plate
 +
 
 +
----
 +
 
 +
=27/08/2010=
 +
 
 +
pilot FACS and Tecan measurements (96-well plate)
 +
 
 +
 
 +
'''[[Igem2010/Main/Measurements/Notebook/September|⇓ September]]'''
 +
 
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Bottom}}
{{:Team:Heidelberg/Bottom}}

Revision as of 01:53, 24 October 2010

Contents

Measurement Standard

⇐ Main Page

⇐ Measurements

08/08/2010

seeding cells for test measurements - 96 well plate for plate-reader and FACS

  • cells were grown in DMEM 10%FBS with phenol red
  • washed with PBS
  • trypsinised (2 ml trypsin)
  • 5ml of OptiMEM media (no FBS) added
  • counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
  • seeded 5000 and 2500 cells/well as on the scheme 96well plate 080810.jpg
  • media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
  • in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids

-> cells did not adhere well, coat plate with poly-L-lysine and repeat

09/08/2010

seeding and transfecting cells for microscopy test measurements

  • add 0.6ul FuGENE reagent to 20ul of OptiMEM
  • mix and incubate 5' at RT
  • add 0.2ug DNA
  • mix and incubate 15' at RT
  • add DNA-FuGENE solution to 10 000 cells (400ul)
  • mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
  • transfere cells to the plate
  • grow 48h


HeLa, EGPF HeLa, EBFP2 HeLa, EGPF and EBFP2 HeLa, EGPF and EBFP2 1:10
HEK T-REx, EGFP HEK T-REx, EBFP2 HEK T-REx, EGPF and EBFP2 HEK T-REx, EGPF and EBFP2 1:10

10/08/2010

coating plates with poly-L-lysine

  • add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
  • leave for 30' in the incubator
  • remove poly-L-lysine solution
  • wash once with PBS

new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously

25/08/2010

seeding cells for pilot FACS and Tecan measurements (96-well plate)

26/08/2010

transfection - 96-well plate

27/08/2010

pilot FACS and Tecan measurements (96-well plate)


⇓ September


Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/Measurement_Standard"