Team:Heidelberg/Notebook/Measurement Standard

From 2010.igem.org

(Difference between revisions)
Line 158: Line 158:
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Pagemiddle}}
how do I get a calendar in here??? anyone??
how do I get a calendar in here??? anyone??
 +
'''[[Igem2010/Main|⇐ Main Page]]'''
 +
 +
 +
{| cellspacing="10" style="color:#c0c0c0"
 +
|-
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/July|<font color="#ffecba">July</font>]]
 +
|- style="background:#4e93a4; color:white"
 +
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 +
|-
 +
|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
 +
|-
 +
|'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''
 +
|-
 +
|'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''
 +
|-
 +
|'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25'''
 +
|-
 +
|'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''||
 +
|-
 +
| colspan="7"| <span style="color:#ffffff">-</span>
 +
|}
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/August|<font color="#ffecba">August</font>]]
 +
|- style="background:#4e93a4; color:white"
 +
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 +
|-
 +
|colspan="6"| ||'''1'''
 +
|-
 +
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''[[Igem2010/Main/Measurements/Notebook/August#08/08/2010|8]]'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/August#09/08/2010/|9]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#10/08/2010|10]]'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
 +
|-
 +
|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''21'''||'''22'''
 +
|-
 +
|'''23'''||'''24'''||'''[[Igem2010/Main/Measurements/Notebook/August#25/08/2010|25]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#26/08/2010|26]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#27/08/2010|27]]'''||'''28'''||'''29'''
 +
|-
 +
|'''30'''||'''31'''||colspan="5"|
 +
|}
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/September|<font color="#ffecba">September</font>]]
 +
|- style="background:#4e93a4; color:white"
 +
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 +
|-
 +
|colspan="2"| ||'''1'''||'''[[Igem2010/Main/Measurements/Notebook/September#02/09/2010|2]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#03/09/2010|3]]'''||'''4'''||'''5'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/September#06/09/2010|6]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#07/09/2010|7]]'''||'''8'''||'''[[Igem2010/Main/Measurements/Notebook/September#09/09/2010|9]]'''||'''10'''||'''11'''||'''12'''
 +
|-
 +
|'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''||'''19'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/September#20/09/2010|20]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#21/09/2010|21]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#22/09/2010|22]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#23/09/2010|23]]'''||'''24'''||'''25'''||'''26'''
 +
|-
 +
|'''27'''||'''28'''||'''29'''||'''30'''||colspan="3"|
 +
|-
 +
| colspan="7"| <span style="color:#ffffff">-</span>
 +
|}
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/October|<font color="#ffecba">October</font>]]
 +
|- style="background:#4e93a4; color:white"
 +
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 +
|-
 +
|colspan="4"| ||'''[[Igem2010/Main/Measurements/Notebook/October#1/09/2010|1]]'''||'''[[Igem2010/Main/Measurements/Notebook/October#2/09/2010|2]]'''||'''3'''
 +
|-
 +
|'''4'''||'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''
 +
|-
 +
|'''11'''||'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''
 +
|-
 +
|'''18'''||'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''
 +
|-
 +
|'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
 +
|-
 +
| colspan="7"| <span style="color:#ffffff">-</span>
 +
|}
 +
|}
 +
{{:Team:Heidelberg/Bottom}}
{{:Team:Heidelberg/Bottom}}

Revision as of 02:01, 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

⇐ Main Page

⇐ Measurements

⇑ August

02/09/2010

  • seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
    • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression
    • problem: forgot to coat the plate, therefore cells did not attach

03/09/2010

File:HeLa pilot GFP BFP2.jpg
FACS pilot experiment of EGFP and EBFP2 expression in Hela P4 cells
File:Huh7 pilot GFP BFP2.jpg
FACS pilot experiment of EGFP and EBFP2 expression in Hek T-Rex cells
File:HEK pilot GFP BFP2.jpg
Tecan pilot experiment of EBFP2 and EGFP expression with in Hela P4 cells‎‎
File:HEK TREx pilot GFP BFP2.jpg
Tecan pilot experiment of EBFP2 and EGFP expression with in Hek T-Rex cells‎‎
  • transfection of cells for fluorescent measurement (EGFP and EBFP2):
  • set-up of the plate:
    • non-transfected cell line
    • each cell line transfected with EGFP control plasmid
    • each cell line transfected with EBFP2 control plasmid
    • each cell line double-transfected with EGFP and EBFP2 control plasmids
    • Results:
      • too little cells have been plated
      • Huh7 cells are hard to transfect
      • expression of EBFP2 is much lower than expression of EGFP





































04/09/2010

  • seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
    • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression

05/09/2010

  • transfection of cells for fluorescent measurement (EGFP and EBFP2):
  • set-up of the plate:
    • non-transfected cell line
    • each cell line transfected with EGFP control plasmid
    • each cell line transfected with EBFP2 control plasmid
    • each cell line double-transfected with EGFP and EBFP2 control plasmids

06/09/2010

  • TECAN and FACS pilot measurements of cells transfected with EGFP and EBFP2

07/09/2010

  • seeding and transfection of HeLa and HEK cells for pilot microscopy measurements:


09/09/2010

  • pilot microscopy measurements:

20/09/2010

  • seeding 2 96-well plates with 5000 cells each well:
    • test whether tuning construct is properly expressing firefly and renilla luciferase and thereby testing whether every part is functional

21/09/2010

  • transfection of both 96-well plates with K1-K8:
    • transfect 50ng of each construct
    • check for promoter strength and functionality of the constructs










22/09/2010

File:22092010promoters.jpg
relative expression units(REU) of firefly luciferase compared to renilla luciferase of the different tuning consstructs without any binding sites for the expressed shRNA10
File:22092010promoters2.jpg
comparison of REU of firefly to renilla luciferase in the different cell lines
  • dual luciferase assay on the 2 96-well plates:
    • all constructs are functional as firefly and renilla luciferase are expressed
    • transfection of construct into Hek t-Rex cells reveals that the CMVTetO2 (construct K4) is also functional as the expression of firefly to renilla counts is nearly zero






23/09/2010

dual luciferase assay

File:23092010promoters.jpg
500px
File:23092010promoters2.jpg
500px
how do I get a calendar in here??? anyone?? ⇐ Main Page


July
MTWTFSS
1234
567891011
12131415161718
19202122232425
262728293031
-
August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031
September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930
-
October
MTWTFSS
123
45678910
11121314151617
18192021222324
25262728293031
-

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