Preparation of Yeast DNA Embedded in Agarose Plugs

Preparation of Yeast DNA Embedded in Agarose Plugs

Anja van Brabant

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

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 0.5ml).

2. Harvest the cells by centrifugation at 1300 x g for 5 min. Wash the cell pellet twice with 50mM EDTA, spinning 5 min at 1300 x g as above.

3. Resuspend the cells in 50mM EDTA to a final concentration of 2 X 10⁹ cells/ml and warm the cell suspension at 45^oC for 5 min. Add an equal volume of 1% low-melt InCert (or SeaPlaque) agarose in 50mM EDTA, also prewarmed to 45^oC . (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^oC (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 6ml of freshly preparedspheroplasting solution. Incubate at 37^oC 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 ofLDS solution. Incubate with gentle shaking at 37^oC for at least 15 min. Remove and add fresh LDS solution. Incubate with gentle shaking at 37^oC 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^oC in six-well dishes with TE, pH8.0, covered with Saran wrap to prevent excessive evaporation. High-molecular-mass DNA will usually remain undegraded for greater than six months.

Refer to the CHEF gel apparatus manual for suggested parameters. To visualize all the yeast chromosomes, I use a switch time ramped from 60-120s, 200V, 24 hours. To obtain better separation of the smaller chromosomes, I 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)

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  To 350 ml H₂O add 93 g Na₂EDTA?H₂O 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.

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LDS Solution

• 1% lithium dodecyl sulfate

• 100mM EDTA

• 10mMTris-HCl, pH8.0

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