FRET-Slide Preparation continued

We keep stocks of 1% SeaPlaque GTG Agarose prepared in S media (1.7 g/l yeast nitrogen base w/o amino acids, 5 g/l ammonium sulfate) in 0.9 ml aliquots in 1.5 ml eppendorf tubes stored in our refrigerator. To prepare the pads a tube of agarose is melted in boiling water, and then equilibrated to 70 C. We prepare a fresh mix of 10X nutrients by combining 500 ul of 40% glucose, 100 ul of 5 mg/ml adenine, 100 ul of 2.5 mg/ml uracil, 100 ul of filter sterilized 10% casamino acids and 100 ul of 1% filter sterilized tryptophan (stored refrigerated). To the tube of agarose is added 90 ul of the 10X nutrients to make the standard SDC medium.

The microscope slide is warmed on the heat block (if the room is cold), and then a 30 ul aliquot of the SDC agarose is pipetted into the center of the slide.