Preparation of Yeast DNA Embedded in Agarose Plugs

adapted from Iadonato, S. P., and A. Gnirke. 1996. RARE-cleavage analysis of YACs. Methods Mol Biol 54: 75-85.

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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.

 

Spheroplasting Solution

40 ml 1M Sorbitol (approx. 1M final conc.)

1.6 ml 0.5M EDTA, pH 8.0 (20mM final)

0.4 ml 1M Tris-HCl, pH7.5 (10mM final)

40 µl 2-mercaptoethanol (14mM final)

0.5 mg/ml Zymolyase 20-T

 

NDS Solution

0.5 M EDTA

10 mM Tris base

1% Sarkosyl

(pH 9.5)

 

To 350 ml H₂O add 93 g Na₂EDTA•2H₂O and 0.6g Tris base

Adjust the pH to greater than 8.0 with 100 to 200 pellets of solid NaOH

Add 50 ml of 10% N-lauryl sarcosine

Adjust the pH to 9.5 with concentrated NaOH

Bring the final volume to 500 ml with H₂O

Filter-sterilize and store at room temperature.

 

LDS Solution

1% lithium dodecyl sulfate

100 mM EDTA

10 mM Tris-HCl, pH 8.0

Per 1 liter:

10 g lithium dodecyl sulfate (Sigma Chemical Cat #L-4632)

200 ml 0.5M EDTA, pH8.0

10 ml 1M Tris-HCl, pH8.0

Bring volume to 1 liter with H₂O, filter-sterilize, and store at room temperature

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