Lab 6: Analyzing Secretion Defects by Western Blotting
Lab 7: Probing and Detecting the Western Blot
Phenotypes of Secretory Mutants
Microsoft Word File: Media:Yeast Spotting Protocol.doc
PROTOCOL - YEAST SPOTTING:
- Obtain 3 sterile plates of Sabauraud’s dextrose media lacking uracil (SD-U) and 3 plates of Sabauraud’s dextrose media lacking uracil and histidine but containing added histidinol (SD-U-H). [Remember that histidinol is the substrate for the His4 enzyme. Supplementing the SD-U-H medium with histidinol pushed the His4 enzymatic reaction in the direction of the product (i.e. histidine), which will produce a much more robust outcome in the reporter assay than if no histidinol was added]. Dry the surface of the plates by turning on the blowers in a disinfected laminar flow hood, placing each plate in the hood with its lid ajar, and leaving the media exposed to the circulating filtered air for long enough to dry the surface but not long enough to dehydrate the plates. 10 minutes should be sufficient.
- While your plates are drying, label 9 sterile microfuge tubes with the names of each of the 9 yeast strains to be tested. See the results chart provided for an abbreviated version of the strain names. Avoid contaminating the lip or inside lid of the microfuge tubes by touching them with your fingers. Although you will not be working in the hood, you should use your best aseptic technique throughout this protocol.
- Place 100 microliters of sterile water into each of the prepared microfuge tubes.
- With a sterile toothpick, transfer 1 large or several small transformed colonies from one strain into the appropriate tube prepared above. Twirl the tip of the toothpick in the water to transfer all the yeast. Discard the toothpick in an autoclave bag. Secure the cap without contaminating it and vortex the water and yeast mixture. Make sure the mixture looks visibly cloudy. If not, add another yeast colony (or several). Repeat for all 9 strains until all cell dilutions seem equally turbid. Vortex well.
- Make a key for spotting each of the 9 yeast strains onto each agar plate on the Media:Yeast Spotting Template.doc. You do not have to label all 3 template circles, but make a key on one template circle and be sure to follow it when you spot all nine strains on each of the 6 plates. There 3 template circles/sheet for the 3 temperatures of incubation. There are 9 diamonds per plate to show where each of the 9 strains will be spotted on each of the 6 plates. At each diamond label a strain name making sure all 9 strains are indicated on the template circle.
- Place the 3 SD-U plates on top of the circles of the SD-U labeled template. Label the bottom (the agar half) of each plate with the following: name of the media (either SD-U or SD-U-H), incubation temperature (either 25, 30, or 37C), your initials, today’s date, and your lab day. Make a mark on the plates where each plate lines up with the template orientation guide. Do the same for the 3 SD-U-HIS plates.
- Following your key, aseptically pipet 5 microliters of each diluted yeast strain prepared in step #4 onto each one of the 6 plates. Be careful to ensure that every plate gets spotted with all 9 strains and that all spots are in the same position on each plate as indicated by your template key. To avoid cross contamination, do not move any of the plates until all the spots have dried thoroughly. When the spots are dry, incubate one plate of each media at each of the 3 temperatures: 25°, 30°, & 37°C for a week.
Assignment 5: Yeast Growth Predictions/BLAST
This should be started during lab time so you can ask any questions you might have.