Difference between revisions of "20.109(F16):Module 1"

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'''Instructors:''' [http://be.mit.edu/directory/noreen-lyell Noreen Lyell], [http://be.mit.edu/directory/leslie-mcclain Leslie McClain] and [http://be.mit.edu/directory/maxine-jonas Maxine Jonas]
 
'''Instructors:''' [http://be.mit.edu/directory/noreen-lyell Noreen Lyell], [http://be.mit.edu/directory/leslie-mcclain Leslie McClain] and [http://be.mit.edu/directory/maxine-jonas Maxine Jonas]
  
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'''TA:''' <br>
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'''TA:''' Emily Clark <br>
 
'''Lab manager:''' Hsinhwa Lee <br>
 
'''Lab manager:''' Hsinhwa Lee <br>
  
 
==Overview==
 
==Overview==
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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.   
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In this module you will measure genomic instability using two techniques: the CometChip and 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.   
  
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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.  
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Next, you will use the CometChip assay to measure DNA damage in response to chemical treatments and to assess repair capacity across different cell lines.  Specifically, you will study the effect of oxidative stress in these experiments.  Last, you will examine the effect of chemical treatment on the abundance of double-strand breaks using an immunofluorescence approach.  
  
  
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[[Image:Fa16 M1 overview.png|thumb|center|700px|Experimental overview for Module 1]]
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[[Image:Fa16 overview v2.png|thumb|center|600px|Experimental overview for Module 1]]
  
 
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<br style="clear:both" />
  
 
==Lab links: day by day==
 
==Lab links: day by day==
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[[20.109(F16):DNA engineering using PCR (Day1)| M1D1: DNA engineering using PCR]]<br>
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[[20.109(F16):Prepare comet chips (Day1)| M1D1: Prepare microwell array and practice tissue culture]]<br>
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[[20.109(F16):Clean and cut DNA (Day2)| M1D2: Clean and cut DNA]]<br>
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[[20.109(F16):Test comet chip loading variables (Day2)| M1D2: Develop experiment to test loading variables and quantify growth rate]]<br>
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[[20.109(F16):Agarose gel electrophoresis (Day3)| M1D3: Agarose gel electrophoresis]]<br>
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[[20.109(F16):Measure DNA damage with comet chip (Day3)| M1D3: Test role of biochemical factors in genomic stability]]<br>
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[[20.109(F16):DNA ligation & transformation (Day4)| M1D4: Ligation & transformation]]<br>
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[[20.109(F16):Asses DNA repair variability with comet chip (Day4)| M1D4: Query inter-individual variability in exposure susceptibility]]<br>
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[[20.109(F16):Examine candidate clones & tissue culture (Day5)| M1D5: Examine candidate clones and tissue culture]]<br>
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[[20.109(F16):Seed cells for immuno-flourescence assay (Day5)| M1D5: Develop approach for sub-nuclear visualization of DNA damage]]<br>
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[[20.109(F16):Lipofection (Day6)| M1D6: Lipofection]]<br>
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[[20.109(F16):Complete immuno-fluorescence assay (Day6)| M1D6: Query DNA repair capacity in tumor cells]]<br>
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[[20.109(F16):FACS analysis (Day7)| M1D7: Data analysis]]<br>
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[[20.109(F16):Data analysis (Day7)| M1D7: Analysis of sub-nuclear foci]]<br>
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==Data==
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See all M1 student data on the [http://engineerbiology.org/wiki/Talk:20.109(F16):Module_1 Discussion page].
  
 
==Assignments==
 
==Assignments==
  
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[[20.109(F16): DNA engineering summary | DNA engineering summary]]<br>
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[[20.109(F16): M1 Data Summary  | Data summary]] <br>
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[[20.109(F16): DNA engineering presentation | DNA engineering mini-presentation]]
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[[20.109(F16): M1 Mini-presentation | Mini-presentation]] <br>
  
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==Useful online resources==
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==References==
  
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*Guidelines for all aspects of [https://www.neb.com/tools-and-resources/usage-guidelines/guidelines-for-pcr-optimization-with-taq-dna-polymerase PCR]
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[[Media:CometChip Jove article.pdf| CometChip: A high-throughput 96-well platform for measuring DNA damage in microarrayed human cells.]] ''Journal of Visualized Experiments'' 92: 1-11.
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*[https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder Double digest buffer identification]
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A video of the procedure can be found [http://www.jove.com/video/50607/cometchip-high-throughput-96-well-platform-for-measuring-dna-damage here].
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*Creation of a [http://nc2.neb.com/NEBcutter2/ restriction map]
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*[http://nebiocalculator.neb.com/#!/ligation Calculator] for buffers and molar ratios
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==References==
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[[Media:CometChip-Pages from Ge 2012 Methods in Cell Bio.pdf| CometChip: Single-cell microarray for high-throughput detection of DNA damage.]] ''Methods in Cell Biology'' 112: 247-268.
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#'''DNA double-strand break repair: From mechanistic understanding to cancer treatment'''<br>''DNA Repair'' 2007<br> Thomas Helleday, Justin Lo, Dik C. van Gent, Bevin P. Engelward<br> [http://dx.doi.org/10.1016/j.dnarep.2007.02.006 URL] <br>[http://web.mit.edu/engelward-lab/animations/DSBR.html Sample Animation] <font color = 000FFF>Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!</font color><br>
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#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://dx.doi.org/10.1016/S0304-419X(01)00018-X URL]
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#'''Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death'''<br>'' EMBO J'' 15 January 1998<br> E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda M <br> [http://doi.org/10.1093/emboj/17.2.598 URL]
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#'''NEBuffer Performance Chart with Restriction Enzymes'''<br>Old buffer system: [https://www.neb.com/~/media/NebUs/Files/nebuffer-performance-chart-with-restriction-enzymes.pdf URL]<br>New buffer system: [https://www.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes URL]
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==Notes for teaching faculty==
 
==Notes for teaching faculty==
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[[20.109(F16): TA notes for module 1| TA notes, M1]]
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[[20.109(F16): TA notes for M1| F16 notes for M1]]
 
[[20.109(F16): TA notes for orientation| F16 notes for orientation day]]
 
[[20.109(F16): TA notes for orientation| F16 notes for orientation day]]

Latest revision as of 13:43, 23 September 2016

20.109(F16): Laboratory Fundamentals of Biological Engineering

Engelward PNAS 2006.png

Schedule Fall 2016        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Module 1

Lecturer: Bevin Engelward
Instructors: Noreen Lyell, Leslie McClain and Maxine Jonas

TA: Emily Clark
Lab manager: Hsinhwa Lee

Overview

In this module you will measure genomic instability using two techniques: the CometChip and 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 treatments and to assess repair capacity across different cell lines. Specifically, you will study the effect of oxidative stress in these experiments. Last, you will examine the effect of chemical treatment on the abundance of double-strand breaks using an immunofluorescence approach.


Experimental overview for Module 1


Lab links: day by day

M1D1: Prepare microwell array and practice tissue culture
M1D2: Develop experiment to test loading variables and quantify growth rate
M1D3: Test role of biochemical factors in genomic stability
M1D4: Query inter-individual variability in exposure susceptibility
M1D5: Develop approach for sub-nuclear visualization of DNA damage
M1D6: Query DNA repair capacity in tumor cells
M1D7: Analysis of sub-nuclear foci

Data

See all M1 student data on the Discussion page.

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 92: 1-11. A video of the procedure can be found here.

CometChip: Single-cell microarray for high-throughput detection of DNA damage. Methods in Cell Biology 112: 247-268.

Notes for teaching faculty

F16 notes for M1

F16 notes for orientation day
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