karthi at deakin.edu.au (Karthi Ramachandran) writes:
>1. My strains are Mat alpha. Is a-factor commercially available or do I have
>to allow them to switch mating types to Mat a and use alpha factor instead.
a-factor is not commercially available to my knowledge. Theoretically,
you could purify a-factor from a-cell conditioned media, but switching mating
types would be much easier.
>2. Do you have a good protocol for synchronising yeast cells. I need to know
>the concentration of the mating harmones used.
Treat MATa cells with 1 microgram per ml alpha-factor (1000X stock =
1 mg/ml in DMSO) for 90 min. Check cells under the microscope for uniform
shmooing. Wash twice[QUICKLY!] with prewarmed 30C media. They're off.
We take samples every 10-20 min for 2 hrs depending on the particular
experiment. You can test the synchrony of the cells by looking at the
budding index (# budded cells/total cells) over the time course.
>3. Our local peptide synthesise facility wants A$ 1500 for 20mg of the alpha
>factor peptide. Is that the normal price or am I being ripped off?
The same amount from Sigma would cost ~US$ 1300. You'll have to do
the math since I don't know the conversion factor. Actually, there are
probably cheaper sources out there, but I don't know them. Anyone?