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==Overview== | ==Overview== | ||
| − | In this module you will measure genomic instability using two techniques: | + | In this module you will measure genomic instability using two techniques: a microwell array (CometChip) and a sub-nuclear foci assay (γH2AX immunofluorescence). Your first task is to critically think through the development of the CometChip assay and determine which conditions provide the best results for loading mammalian cells into the device. To this end, you will consider variables that affect cell loading into the microwells of the CometChip. The data you collect will be used to determine the conditions for subsequent assays. |
| − | Next, you will use the CometChip assay to measure DNA damage in response to chemical | + | Next, you will use the CometChip assay to measure DNA damage in response to a deficiency in repair and a chemical treatment. Specifically, you will study the role of DNA-pk in DNA repair and the effects of oxidative stress on DNA damage via H<sub>2</sub>O<sub>2</sub> treatment. Then, you will use the γH2AX to visualize DNA damage in individual cells and compare the results to the CometChip data. |
| − | [[Image: | + | [[Image:Fa18 M1 overview.png|thumb|center|650px]] |
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Revision as of 17:33, 4 September 2018
Contents
Module 1
Lecturer: Bevin Engelward
Instructors: Noreen Lyell, Leslie McClain and Josephine Bagnall
TAs: Corban Swain and Jai Padmakumar
Lab manager: Hsinhwa Lee
Overview
In this module you will measure genomic instability using two techniques: a microwell array (CometChip) and a sub-nuclear foci assay (γH2AX immunofluorescence). Your first task is to critically think through the development of the CometChip assay and determine which conditions provide the best results for loading mammalian cells into the device. To this end, you will consider variables that affect cell loading into the microwells of the CometChip. The data you collect will be used to determine the conditions for subsequent assays.
Next, you will use the CometChip assay to measure DNA damage in response to a deficiency in repair and a chemical treatment. Specifically, you will study the role of DNA-pk in DNA repair and the effects of oxidative stress on DNA damage via H2O2 treatment. Then, you will use the γH2AX to visualize DNA damage in individual cells and compare the results to the CometChip data.
Lab links: day by day
M1D1:
M1D2:
M1D3:
M1D4:
M1D5:
M1D6:
M1D7:
Assignments
Data summary
Mini-presentation
References
CometChip: A high-throughput 96-well platform for measuring DNA damage in microarrayed human cells. Journal of Visualized Experiments. (2014) 92: 1-11.
- A video of the procedure is linked here.
CometChip: Single-cell microarray for high-throughput detection of DNA damage. Methods in Cell Biology. (2012) 112: 247-268.
