20.109(F14):Module 1: Difference between revisions
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(New page: {{Template:20.109(F14)}} <div style="padding: 10px; width: 640px; border: 5px solid #66399F;"> ==Module 1== '''Instructors:''' [http://web.mit.edu/be/people/engelward.shtml Bevin Engelw...) |
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==Module 1== | ==Module 1== | ||
'''Instructors:''' [http://web.mit.edu/be/people/engelward.shtml Bevin Engelward], [[User:Shannon K. Alford |Shannon Hughes]], [[User: | '''Instructors:''' [http://web.mit.edu/be/people/engelward.shtml Bevin Engelward], [[User:Shannon K. Alford |Shannon Hughes]], [[User:AgiStachowiak| Agi Stachowiak]] and [[User: Noreen L. Lyell | Noreen Lyell]] | ||
'''TA:''' <br> | '''TA:''' [[User:Isaak E. Mueller | Isaak Mueller]] <br> | ||
In this experimental module you will modify the gene for ''EGFP'' (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment. | In this experimental module you will modify the gene for ''EGFP'' (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment. | ||
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[[20.109(F14): Mod 1 Day 2 Clean and cut DNA | Day 2: Clean and cut DNA]]<br> | [[20.109(F14): Mod 1 Day 2 Clean and cut DNA | Day 2: Clean and cut DNA]]<br> | ||
[[20.109(F14): Mod 1 Day 3 Agarose gel electrophoresis| Day 3: Agarose gel electrophoresis]]<br> | [[20.109(F14): Mod 1 Day 3 Agarose gel electrophoresis| Day 3: Agarose gel electrophoresis]]<br> | ||
[[20.109(F14): Mod 1 Day 4 DNA | [[20.109(F14): Mod 1 Day 4 DNA Ligation & Transformation| Day 4: Ligation & Transformation]]<br> | ||
[[20.109(F14): Mod 1 Day 5 Examine candidate clones & tissue culture| Day 5: Examine candidate clones | [[20.109(F14): Mod 1 Day 5 Examine candidate clones & tissue culture| Day 5: Examine candidate clones and Tissue Culture]]<br> | ||
[[20.109(F14): Mod 1 Day 6 Lipofection | [[20.109(F14): Mod 1 Day 6 Lipofection | Day 6: Lipofection]]<br> | ||
[[20.109(F14): Mod 1 Day 7 FACS analysis| Day 7: | [[20.109(F14): Mod 1 Day 7 FACS analysis| Day 7: Data analysis]]<br> | ||
==Assignments== | ==Assignments== | ||
Abstract and data summary: [[20.109( | Abstract and data summary: [[20.109(F14): DNA engineering summary | Assignment description]] | ||
Plasmid construction methods section: [[20.109( | Plasmid construction methods section: [[20.109(F14): DNA engineering methods | Assignment description]] | ||
==References== | ==References== | ||
#'''Single cell trapping and DNA damage analysis using microwell arrays'''<br>'' PNAS'' 1 June 2010<br> D K Wood, D M Weingeist, S N Bhatia, B P Engelward<br> [http://www.pnas.org/content/early/2010/05/12/1004056107 URL] | |||
#'''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 = 9900CC>Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!</font color><br> | #'''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 = 9900CC>Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!</font color><br> | ||
#'''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] | #'''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] | ||
#'''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] | #'''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] | ||
#'''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] | #'''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] | ||
==Notes for Teaching Faculty== | ==Notes for Teaching Faculty== | ||
[[20.109( | [[20.109(F14): TA notes for module 1| TA notes, mod 1]] | ||
[[20.109( | [[20.109(F14): TA notes for orientation| F14 notes for orientation day]] | ||
Latest revision as of 10:16, 11 September 2014
Module 1
Instructors: Bevin Engelward, Shannon Hughes, Agi Stachowiak and Noreen Lyell
TA: Isaak Mueller
In this experimental module you will modify the gene for EGFP (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP in vivo. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event. We will then choose a few variables to test on the final day of the experiment.
Lablinks: day by day
Day 1: DNA engineering using PCR
Day 2: Clean and cut DNA
Day 3: Agarose gel electrophoresis
Day 4: Ligation & Transformation
Day 5: Examine candidate clones and Tissue Culture
Day 6: Lipofection
Day 7: Data analysis
Assignments
Abstract and data summary: Assignment description
Plasmid construction methods section: Assignment description
References
- Single cell trapping and DNA damage analysis using microwell arrays
PNAS 1 June 2010
D K Wood, D M Weingeist, S N Bhatia, B P Engelward
URL - 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
