Team:Heidelberg/Notebook/Measurement Standard

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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