Ligation & Transformation Creating Transgenics Ligation & Transformation

Ligation Ligase: an enzyme which connects sections of DNA This graphic is from yesterday’s PowerPoint. From http://library.thinkquest.org/04apr/00217/en/products/recdna/about.html

Ligation Ligase: an enzyme which connects sections of DNA Materials: DNA from yesterday A few pieces of tape Worksheet Goal: create bacterial plasmids that contain a jellyfish or coral gene Match sections of your “sticky ends” that are complementary. Tape models ligase: ligate the sections together Do the front of the worksheet

Transgenic Containing DNA from multiple species Are your plasmids transgenic?

Transformation = Inserting foreign genes into bacteria Requires small, circular “plasmids” of DNA

Transformation Protocol Treat bacteria with CaCl2 in order to permeabilize their cell wall. Keeping bacteria on ice, add plasmid to the bacteria. For exactly 45 seconds, incubate the bacteria at 42ºC to create ‘holes’ in the cell wall that let the plasmid in. Put the bacteria back on ice, to keep from killing them (in heat) and to seal in the plasmids they’ve absorbed. Grow the bacteria, so they can make the protein!

Transformation Efficiency How many of our bacteria actually got transformed? It’s typical for a tiny percent of the bacteria to successfully take in the plasmid, but you only need one…

Selectable Markers Problem: most of the bacteria do not have our gene. They’ll overwhelm the bacteria that were transformed! We need a way to grow only those bacteria that successfully took in the plasmid. The plasmid includes a “selectable marker”: only those bacteria that have the plasmid can survive!

Pictures from: Tsien Lab: http://ucsdnews.ucsd.edu/newsrel/science/tsien/ 4 plates: http://www.flickr.com/photos/imagined_horizons/4327903307/ 6 plates: http://www.ebbep.org/