BE.109:DNA engineering - Revision history

AgiStachowiak: Undo revision 157507 by Special:Contributions/Bevin (User talk:Bevin)

2007-10-10T17:32:50Z

Undo revision 157507 by Special:Contributions/Bevin (User talk:Bevin)

←Older revision		Revision as of 17:32, 10 October 2007
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	==Module 1==		==Module 1==

-	'''Instructors:''' ~~[http://web.mit.edu/be/people/engelward.htm~~ Bevin Engelward] and [[Natalie Kuldell]]	+	'''Instructors:''' Bevin Engelward and [[Natalie Kuldell]]

-	'''TA:''' [[~~David Weingeist~~]]	+	'''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.		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.

Bevin: /* Module 1 */

2007-10-10T17:02:16Z

Module 1

←Older revision		Revision as of 17:02, 10 October 2007
Line 6:		Line 6:
	==Module 1==		==Module 1==

-	'''Instructors:''' Bevin Engelward and [[Natalie Kuldell]]	+	'''Instructors:''' [http://web.mit.edu/be/people/engelward.htm Bevin Engelward] and [[Natalie Kuldell]]

-	'''TA:''' [[~~Yoon Sung Nam~~]]	+	'''TA:''' [[David Weingeist]]

	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.

Reshma P. Shetty: /* References */

2006-10-09T19:57:46Z

References

←Older revision		Revision as of 19:57, 9 October 2006
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	'''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.		'''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<br> Thomas Helleday<br> [http://~~openwetware~~.org/~~images~~/~~9/99/HR_Review_Helleday03~~.~~pdf PDF reprint~~]	+	#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://dx.doi.org/10.1016/j.mrfmmm.2003.08.013 URL]
-	#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://~~www~~.~~sciencedirect~~.~~com~~/~~science?_ob=MImg&_imagekey=B6T23~~-~~42HNM0T~~-~~3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint~~]	+	#'''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<br> [http://www.pubmedcentral.gov/picrender.fcgi?artid=1170409&blobtype=pdf PDF reprint]		#'''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<br> [http://www.pubmedcentral.gov/picrender.fcgi?artid=1170409&blobtype=pdf PDF reprint]

Yoonsung: /* References */

2006-02-14T04:07:33Z

References

←Older revision		Revision as of 04:07, 14 February 2006
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	#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]		#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]
	#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]		#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]
		+	#'''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<br> [http://www.pubmedcentral.gov/picrender.fcgi?artid=1170409&blobtype=pdf PDF reprint]

Yoonsung: /* References */

2006-02-10T02:28:17Z

References

←Older revision		Revision as of 02:28, 10 February 2006
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	#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]		#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]
	#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]		#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]
-	~~[[Schiestl.pdf\|Schiestl]]~~

Yoonsung: /* References */

2006-02-10T02:27:59Z

References

←Older revision		Revision as of 02:27, 10 February 2006
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	#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]		#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]
	#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]		#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]
-	[[~~Image:~~Schiestl.pdf\|Schiestl]]	+	[[Schiestl.pdf\|Schiestl]]

Yoonsung: /* References */

2006-02-10T02:27:15Z

References

←Older revision		Revision as of 02:27, 10 February 2006
Line 62:		Line 62:
	#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]		#'''Pathways for mitotic homologous recombination in mammalian cells'''<br>''Mutation Research'' 27 November 2003<br> Thomas Helleday<br> [http://openwetware.org/images/9/99/HR_Review_Helleday03.pdf PDF reprint]
	#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]		#'''Homologous recombination as a mechanism of carcinogenesis'''<br>'' Biochim Biophys Acta'' 21 March 2001<br> Bishop AJ and Schiestl RH<br> [http://www.sciencedirect.com/science?_ob=MImg&_imagekey=B6T23-42HNM0T-3-7&_cdi=4907&_user=501045&_orig=search&_coverDate=03%2F21%2F2001&_qd=1&_sk=985289996&view=c&wchp=dGLbVzz-zSkzV&md5=d3f3f0cc3eb1bb8b91a07fba5daebf5c&ie=/sdarticle.pdf PDF reprint]
		+	[[Image:Schiestl.pdf\|Schiestl]]

Yoonsung: /* DNA engineering web links */

2006-02-10T02:26:40Z

DNA engineering web links

←Older revision		Revision as of 02:26, 10 February 2006
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	Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/		Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/

-	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc/\|pCX-EGFP]]	+	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc\|pCX-EGFP]]

	ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html		ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html

Yoonsung: /* DNA engineering web links */

2006-02-10T02:25:53Z

DNA engineering web links

←Older revision		Revision as of 02:25, 10 February 2006
Line 46:		Line 46:
	Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/		Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/

-	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc\|pCX-EGFP]]	+	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc/\|pCX-EGFP]]

	ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html		ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html

Yoonsung: /* DNA engineering web links */

2006-02-10T02:25:16Z

DNA engineering web links

←Older revision		Revision as of 02:25, 10 February 2006
Line 46:		Line 46:
	Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/		Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/

-	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc\|pCX-EGFP~~.doc~~]]	+	pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf [[Image:Macintosh_HD-Users-nkuldell-Desktop-pCX-EGFP.doc\|pCX-EGFP]]

	ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html		ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html