20.109(F11):Module 1

Module 1
Instructors: Bevin Engelward, Natalie Kuldell, Agi Stachowiak

TA:

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: DNA ligation and bacterial transformation Day 5: Examine candidate clones & introduction to tissue culture Day 6: Lipofection & lab practical Day 7: FACS analysis

DNA engineering lab practical DNA engineering powerpoint summary

Notes for Teaching Faculty
TA notes, mod 1