Preparation of Yeast DNA Embedded in Agarose Plugs
互联网
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. )
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 45o C for 5 min. Add an equal volume of 1% low-melt InCert (or SeaPlaque) agarose in 50mM EDTA, also prewarmed to 45o 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 4o C (for 15 minutes).
spheroplasting solution . Incubate at 37o 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 37o C for at least 15 min. Remove and add fresh LDS solution. Incubate with gentle shaking at 37o 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 4o C 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
To 350 ml H2 O add 93 g Na2 EDTA?H2 O and 0.6g Tris base
- 0.5 M EDTA
- 10 mM Tris base
- 1% Sarkosyl
- (pH 9.5)
- 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 H2 O
- Filter-sterilize and store at room temperature.
- 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 H2 O, filter-sterilize, and store at room temperature
