BE.109:DNA engineering

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 2: Clean and cut DNA

 Day 3: Agarose gel electrophoresis

 Day 4: DNA ligation and bacterial transformation

 Day 5: Examine candidate clones

 Day 6: Restriction map and tissue culture

 Day 7: Lipofection

 Day 8: FACS analysis

 Module 1 lab report schedule and guidelines 

DNA engineering web links
Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/

pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf

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