BE.109:DNA engineering: Difference between revisions
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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. | ||
[[Image:Be109recombomouse.jpg|thumb|left|500px|Recombocell Image from Dominika Wiktor of the Engelward Lab''']] | [[Image:Be109recombomouse.jpg|thumb|left|500px|'''Recombocell Image from Dominika Wiktor of the Engelward Lab''']] | ||
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'''[[BE.109: Schedule for your Mod1 Lab Report]]''' | '''[[BE.109: Schedule for your Mod1 Lab Report | Module 1 lab report schedule]]''' | ||
==DNA engineering web links== | ==DNA engineering web links== | ||
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==References== | ==References== | ||
'''Note:''' PDF reprints are provided below within the context of [http://www.copyright.gov/fls/fl102.html fair use]. Please obtain copies from the publisher if appropriate. | |||
#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003 DOI:10.1016/j.mrfmmm.2003.08.013<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint] |
Revision as of 17:27, 8 February 2006
Module 1
Instructors: Bevin Engelward and Natalie Kuldell
TA: Yoon Sung Nam
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.
Lab handouts
Day 1: DNA engineering using PCR (you will also need weblinks, below)
Day 3: Agarose gel electrophoresis
Day 4: DNA ligation and bacterial transformation
Day 5: Examine candidate clones
Day 6: Restriction map and tissue culture
DNA engineering web links
Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/
pCX-EGFP plasmid map: File:Macintosh HD-Users-nkuldell-Desktop-pCX-EGFP.doc
ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html
NCBI: http://www.ncbi.nlm.nih.gov/
Cybergene: http://www.cybergene.se/primer.html
New England Biolabs: http://www.neb.com/nebecomm/default.asp
References
Note: PDF reprints are provided below within the context of fair use. Please obtain copies from the publisher if appropriate.
- Pathways for mitotic homologous recombination in mammalian cells
Mutation Research 27 November 2003 DOI:10.1016/j.mrfmmm.2003.08.013
Thomas Helleday
PDF reprint