PROTOCOLS FOR EXPERIMENT 5 (Labs 8, 9, 10 and 11)

Lab 8: Restreaking the mutants

Every student will have their own plates.

Pick and restreak 8 red colonies. The point of this is to streak to single colonies. These are the strains that you will work with for the rest of the experiment.

1.) You will be given 2 MM+ his trp ade and can plates. Using a marker divide the plate into four quadrants.

2.) Flame the loop and cool in the plate. It is important that the loop is cool enough so that you do not fry your yeast cells.

3.) Using the flamed loop pick a colony and spread across the top of the plate.

4.) Flame the loop and cool in the plate. Pull down toward the center of the plate.

5.) Flame the loop and cool in the plate. Streak across the second line. Repeat until you have restreaked eight mutants.

6.) Put the plates in the 30°C incubator with the plates facing down (in other words, upside down).

7.) In lab 9, note the colour of each strain in your notebook.

Lab 9: Yeast mating for complementation experiment

You will be given a swi5 mutant tester strain and the unmutagenized parental strain.

1.) Draw nine squares on the bottom YEPD plate with a marker.

2.) Spread the swi5 tester strain in to each of the nine squares.

3.) Pick a mutant and mix in one of the nine squares, repeat until all eight strains are placed on the plate. In the ninth square mate the swi5 tester strain with the parental strain; this is a control mating.

4.) Put the plates in the 30°C incubator facing down.

Lab 10: Complementation analysis and Beta-glactosidase assay

A) Complementation analysis

1.) Make nine squares on the MM + his and ade plate.

2.) Spread some of the mated strains from last week in each of the nine squares of your YEPD plate.

3.) Put the plates in the 30°C incubator facing down.

4.) In lab 11 note which are red and which are white.

B) Beta-galactosidase assay

1.) Draw nine squares on the bottom of the MM+ Galactose, ade, his, trp plate.

2.) Place a piece of filter paper on the plate. Note the orientation of the filter paper for later reference.

3.) Spread each of your eight strains FROM YOUR ORIGINAL STREAKED MUTANT PLATE (lab 8) in a square and into the ninth spread some of the parental strain.

4.) Put the plates in the 30°C incubator facing down.

Lab 11: Beta-galactosidase assay (con't)

1.) Using a pair of forceps take off the filter paper from the plate.

2.) Submerge or float the filter yeast side down in liquid nitrogen for 5-10 seconds.

3.) Place cells side up in lid of Petri dish. Pipette 2 mL of Z-buffer onto the filter paper being sure to saturate it.

Z-buffer:60mM Na2HPO4, 40mM NaH2PO4; 10mM KCl; 1mM MgSO4; 2-mercaptoethanol; 1mg/ml X-Gal

4.) Let the colour reaction develop at 30°C.

Record data: Take a picture using the digital camera provided. Make sure that your group number and initials are included somewhere in the picture.