20.109(F16):Module 1
Contents
Module 1
Lecturer: Bevin Engelward
Instructors: Noreen Lyell, Leslie McClain and Maxine Jonas
TA:
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 assess the base excision repair pathway and the nucleotide excision repair 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: DNA engineering using PCR
M1D2: Clean and cut DNA
M1D3: Agarose gel electrophoresis
M1D4: Ligation & transformation
M1D5: Examine candidate clones and tissue culture
M1D6: Lipofection
M1D7: Data analysis
Assignments
DNA engineering summary
DNA engineering mini-presentation
Useful online resources
- Guidelines for all aspects of PCR
- Double digest buffer identification
- Creation of a restriction map
- Calculator for buffers and molar ratios
References
- DNA double-strand break repair: From mechanistic understanding to cancer treatment
DNA Repair 2007
Thomas Helleday, Justin Lo, Dik C. van Gent, Bevin P. Engelward
URL
Sample Animation Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!
- Homologous recombination as a mechanism of carcinogenesis
Biochim Biophys Acta 21 March 2001
Bishop AJ and Schiestl RH
URL - Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
EMBO J 15 January 1998
E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda M
URL - NEBuffer Performance Chart with Restriction Enzymes
Old buffer system: URL
New buffer system: URL