Preparation of Yeast DNA Embedded in Agarose Plugs
Preparation of Yeast DNA Embedded in Agarose Plugs
Anja van Brabant
(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 45oC for 5 min. Add an equal volume of 1% low-melt InCert (or SeaPlaque) agarose in 50mM EDTA, also prewarmed to 45oC . (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 4oC (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 37oC 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 37oC for at least 15 min. Remove and add fresh LDS solution. Incubate with gentle shaking at 37oC 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 4oC 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.
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
0.5 M EDTA
10 mM Tris base
1% Sarkosyl
(pH 9.5)
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.
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
