20.109(F16):Module 1
Contents
Module 1
Lecturer: Bevin Engelward
Instructors: Noreen Lyell, Leslie McClain and Maxine Jonas
TA: Molly Bird
Lab manager: Hsinhwa Lee
Overview
In this module you will measure DNA repair using two assays: the comet chip and immuno-fluorescence. Your first task is to critically think through the development of the comet chip assay and determine which conditions provide the best results. To this end, you will consider variables that effect cell loading into the microwells of the comet chip and variables that effect the readout of the results. The data you collect will be used to propose a high-throughput comet chip assay for commercial use.
Next, you will use the comet chip assay to assess the effect of chemicals and ultraviolet-irradiation on DNA repair. Specifically, you will study the base excision repair (BER) pathway and the nucleotide excision repair (NER) pathway. Last, you will examine the effect of gamma-irradiation on double strand breaks using an immuno-fluorescence approach.
Lab links: day by day
M1D1: Prepare comet chips
M1D2: Test comet chip loading variables
M1D3: Measure DNA damage with comet chip
M1D4: Assess DNA repair variability with comet chip
M1D5: Seed cells for immuno-flourescence assay
M1D6: Complete immuno-fluorescence assay
M1D7: Data analysis
Assignments
Data summary
Mini-presentation
References
paper for non-htp system
CometChip: Single-cell microarray for high-throughput detection of DNA damage. Methods in Cell Biology 112: 247-268.