A. Making Spheroplasts
1. Harvest Saccharomyces cerevisiae cells from a 25 ml culture by centrifugation at 1000 to 1500 X g for 2 min (see protocol for growth of yeast cells). Discard supernatant.
2. Wash the cells: Add an equal volume of 1 M Sorbitol to the cell pellet, centrifuge again, and discard Sorbitol supernatant.
3. Resuspend cell pellet in 300 μl of SCEM Buffer and transfer to a microcentrifuge.
4. Add 30 μl of 100 mg/ml Novozyme 234 (Novo Enzyme Products) in SCE buffer.
5. Incubate at room temperature for 1 hr.
B. Chromosome preparation
1. After preparing Spheroplasts, centrifuge cells at 3000 rpm for 5 sec in a microcentrifuge and aspirate the supernatant.
2. Resuspend cells in 100 μl of 1 M Sorbitol.
3. Add 100 μl of 1% InCert Agarose solution preincubated at 50°C.
4. Mix gently and pipette into 100 μl gel plug moulds. Place moulds on ice for 5 min to solidify.
5. After the agarose has solidified, place the agarose blocks into 1 ml of Proteinase K Solution and incubate at 50°C for at least 4 hrs.
6. After first incubation, remove the Proteinase K Solution and add another 1 ml of fresh Proteinase K Solution. Incubate overnight at 50°C.
7. After second incubation, remove solution and sequentially wash the blocks in the following order:
a. In 1 ml of TE/PMSF, four times 30 min. incubations with mixing (i.e. on a nutator),
b. In 1 ml of TE, four times 30 min. incubations with mixing,
c. In 1 ml of 0.5X TE, two times 30 min. with mixing.
Store blocks at 4°C.
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