Team:Heidelberg/Notebook/miMeasure/August

From 2010.igem.org

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seeding cells for test measurements - 96 well plate for plate-reader and FACS
seeding cells for test measurements - 96 well plate for plate-reader and FACS
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*cells were grown in DMEM 10%FBS with phenol red
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test of media:
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*washed with PBS
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*OptiMEM 0% FBS + Pen/Strep + L-Glu + other according to cell line
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*trypsinised (2 ml trypsin)
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*OptiMEM 2% FBS + Pen/Strep + L-Glu + other according to cell line
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*5ml of OptiMEM media (no FBS) added
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*DMEM 10% FBS+ Pen/Strep + L-Glu + other according to cell line
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*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
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*seeded 5000 and 2500 cells/well as on the scheme [[96well plate 080810.jpg]]
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*media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
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*in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids
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-> cells did not adhere well, coat plate with poly-L-lysine and repeat
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* cells did not adhere properly so we decided to coat 96-well plate with poly-L-lysine and repeat
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* significantly better growth in DMEM with 10% FBS, so we decided to grow cells in it and before measurement to change it to OptiMEM, to reduce high background fluorescence coming from FBS.
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===09/08/2010===
===09/08/2010===

Revision as of 08:54, 26 October 2010

Contents

08/08/2010

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

test of media:

  • OptiMEM 0% FBS + Pen/Strep + L-Glu + other according to cell line
  • OptiMEM 2% FBS + Pen/Strep + L-Glu + other according to cell line
  • DMEM 10% FBS+ Pen/Strep + L-Glu + other according to cell line


  • cells did not adhere properly so we decided to coat 96-well plate with poly-L-lysine and repeat
  • significantly better growth in DMEM with 10% FBS, so we decided to grow cells in it and before measurement to change it to OptiMEM, to reduce high background fluorescence coming from FBS.


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


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)

August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031
September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930 -
October
MTWTFSS
123
45678910
11121314151617
18192021222324
25262728293031

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