Preparation of Yeast DNA Embedded in Agarose Plugs

互联网2013-09-06

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1.	Inoculate a 5 ml culture with a single colony from a YAC-containing strain of yeast and grow until saturated. Determine the number of yeast cells per milliliter. The cell count should be roughly 1 x 10⁸ cells/ml for a saturated culture. A 5 ml saturated culture will yield one plug at approximately 5 x 10⁸ cells per plug. (Each plug holds slightly less than 0.5 ml).
2.	Harvest the cells by centrifugation at 1300 x g for 5 min. Wash the cell pellet twice with 50 mM EDTA, spinning 5 min at 1300 x g as above.
3.	Resuspend the cells in 50 mM EDTA to a final concentration of 2 x 10⁹ cells/ml and warm the cell suspension at 45°C for 5 min. Add an equal volume of 1% low-melt InCert (or SeaPlaque) agarose in 50 mM EDTA, also prewarmed to 45°C . (This procedure will make 0.5% plugs, which are fairly fragile, but are better if further manipulations like in gelo digest are required. If not, use 2% agarose to make 1% plugs, which are less fragile). Mix the suspension thoroughly by vortexing and pipette 500 µl aliquots into each plug mold to harden. A 100 ml culture should yield approximately 20 plugs. Plugs may be allowed to set at room temperature or placed at 4°C (for 15 minutes).
4.	Extrude each plug from the plug mold into a six-well dish, up to 3 plugs per well. To each well add 6 ml of freshly prepared spheroplasting solution. Incubate at 37°C for 2-4 h with gentle shaking. Longer incubation times are preferred.
5.	Aspirate off the spheroplast solution from each well and add 6 ml of LDS solution. Incubate with gentle shaking at 37°C for at least 15 min. Remove and add fresh LDS solution. Incubate with gentle shaking at 37°C overnight.
6.	Wash the plugs 3 x 30 min with 6 ml of 0.2x NDS solution with gentle shaking at room temperature.
7.	Wash the plugs 5 x 30 min with 6 ml of TE, pH 8.0 with gentle shaking at room temperature. Plugs may be stored at 4°C in six-well dishes with TE, pH 8.0, covered with Saran wrap to prevent excessive evaporation. High-molecular-mass DNA will usually remain undegraded for at least six months. Refer to the CHEF gel apparatus manual for suggested parameters. To visualize all the yeast chromosomes, we use a switch time ramped from 60-120s, 200V, 24 hours. To obtain better separation of the smaller chromosomes, we use a switch time ramped from 35-70s, 200V, 22 hrs.