BE.109:DNA engineering: Difference between revisions
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'''Instructors:''' Bevin Engelward and [[Natalie Kuldell]] | '''Instructors:''' Bevin Engelward and [[Natalie Kuldell]] | ||
'''TA:''' Yoon Sung Nam | '''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 examine the frequency of recombination when the C-terminally truncated version is “damaged” with different double-strand breaks. | 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 examine the frequency of recombination when the C-terminally truncated version is “damaged” with different double-strand breaks. | ||
Revision as of 10:39, 8 December 2005
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 examine the frequency of recombination when the C-terminally truncated version is “damaged” with different double-strand breaks.
Lab handouts
Day 3: Agarose gel electrophoresis
Day 4: DNA ligation and bacterial transformation
Day 5: Examine candidate clones
