IGEM:IMPERIAL/2009/Assays Protocols/M3 Assays: Difference between revisions
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=Assays for Cell Death Strategy= | =Assays for Cell Death Strategy= | ||
== | ==FACS (Fluorescent Automated Cell Sorter)== | ||
[[Media:Cell Death Assay.pdf]] <b>Qualitative and quantitative</b> | [[Media:Cell Death Assay.pdf]] <b>Qualitative and quantitative</b> | ||
*This is an in vitro assay | *This is an in vitro assay | ||
==Using GFP marker== | |||
<i>Basic genetic circuit for the restriction enzyme testing construct: </i> | <i>Basic genetic circuit for the restriction enzyme testing construct: </i> | ||
| Line 21: | Line 19: | ||
iii) Initial increase in fluorescence, followed by a plateauing of the fluorescence, as the restriction enzymes destroy the genetic material within the cell. | iii) Initial increase in fluorescence, followed by a plateauing of the fluorescence, as the restriction enzymes destroy the genetic material within the cell. | ||
==By Colony Forming Units== | |||
1) Plating dilution series on TSB Agar Plates <br> | 1) Plating dilution series on TSB Agar Plates <br> | ||
2) Counting of colonies after overnight incubation <br> | 2) Counting of colonies after overnight incubation <br> | ||
3) Determining the number of colony forming units (number of cells still alive) <br> | 3) Determining the number of colony forming units (number of cells still alive) <br> | ||
Revision as of 03:30, 4 August 2009
Assays for Cell Death Strategy
FACS (Fluorescent Automated Cell Sorter)
Media:Cell Death Assay.pdf Qualitative and quantitative
- This is an in vitro assay
Using GFP marker
Basic genetic circuit for the restriction enzyme testing construct:
Here, there are 3 possibilities for the expression of the GFP, and these are shown below:
i) Non-functional restriction enzyme co-expressed with the GFP.
ii) Basal levels of GFP expression suggesting that the promoter is not functioning properly.
iii) Initial increase in fluorescence, followed by a plateauing of the fluorescence, as the restriction enzymes destroy the genetic material within the cell.
By Colony Forming Units
1) Plating dilution series on TSB Agar Plates
2) Counting of colonies after overnight incubation
3) Determining the number of colony forming units (number of cells still alive)
