Purpose:

• To isolate intact, high molecular weight DNA from yeast cells for subcloning and rare cutting restriction enzyme analysis. One can expect a yield of 100-200 礸 of DNA per prep.

Time required:

• 6 days total

Day 1-3: 10 minutes
Day 4: 6-8 hours
Day 5: 2 hours
Day 6: 1 hour

Special Equipment:

• 23 mm dialysis tubing

• Beckman SW28 Rotor and Ultracentrifuge

Special Reagents:

• SCE solution

• Lyticase (Sigma # L 8137)

• Yeast lysis buffer

• 15%, 20%, and 50% sucrose solutions

Procedure:

Days 1-3

1. Inoculate a single yeast colony into 5-7 ml of AHC medium and incubate at 30 degrees C for 2 days. Inoculate 250-500 ml of YPD medium with the culture, and incubate with shaking at 30 degrees C overnight.

Day 4

1. Pour the culture volume into large centrifuge bottles and centrifuge at 2500 rpm for 15 minutes in the Beckman J-6 centrifuge. Resuspend the cell pellet in 40 ml of dH2O by pipetting up and down while scraping the cell pellet. Transfer to 50 ml conical tube, and centrifuge at 2500 rpm for 15 minutes; decant supernatent. At this point cells can be frozen for future use, or one can proceed as follows.

2. Resuspend pellet in 3.5 ml of SCE with 30 祃 2-Mercaptoethanol (or 40 祃 2M DTT) and 10 mg Lyticase. Spheroplast at 37 degrees C for 1 or more hours and shake gently every 15 minutes. Continue until the suspension has greatly increased in viscosity (often one can see many small bubbles within the viscous suspension at this point).

3. Pour the spheroplast suspension slowly down the side of a 250 ml flask (used for the large surface area they provide) containing 7 ml of Lysis buffer with 100 礸/ml Proteinase K. Take care to gently mix the solution and achieve uniformity. Place flask in a 65欳 water bath for 15 minutes with occasional shaking, then rapidly cool to room temperature in a water bath.

4. Make a crude sucrose gradient by first adding 11 ml of 20% sucrose, then 11 ml of 15% sucrose, then carefully underlaying 3 ml of 50% sucrose in a 36 ml Beckman centrifuge tube (Ultra-Clear 25x89 mm). Slowly pour the lysate into the tube, then ultracentrifuge in the SW28 rotor at 26,000 rpm at 20欳 for three hours.

5. Aspirate the top of the gradient (approximately 30 ml) until the viscous DNA on the bottom can be seen to move. Collect this bottom layer using a 10 ml pipette, and place in dialysis tubing (23 mm) that has been previously prepared (see section in manual on preparation of dialysis tubing). Attempt to minimize the total volume, for fluid will be taken up into the bag, diluting the DNA concentration. Use rubber bands on the clips to prevent them from opening unexpectedly. Place tubing in a 2 liter flask containing 1 liter of TE, add a stirring bar, and place on a stir plate at 4 degrees C overnight.

Days 5-6

1. Remove dialysis bags and place on a bed of dry sucrose, then cover bags completely with sucrose. When the sucrose becomes damp, replace it with dry sucrose; gradually place the clips nearer to one another on the dialysis tubing, in order to concentrate the DNA. This process should take 30-90 minutes. Dialyze the bags again in TE over the day, then change the TE and allow to dialyze again overnight.

2. Take off the upper clip and place this portion of the dialysis tubing within a 5 ml snap-cap tube. Drain the liquid into the tube. It is very important from this point on to try to minimize shearing of this high molecular weight DNA; pipetting should be kept to a minimum and should be done only using tips which have had their ends cut off. One can expect a yield of 100-200 礸 of DNA per gradient.

3. Check the concentration of the DNA by running against known standards. DNA can be reprecipitated by adding 1/10 volume 3M sodium acetate and 2 volumes of cold 95% ethanol, then gently spooling out DNA; if done carefully, little shearing will occur. Add RNase at 50 礸/ml when using the DNA in restriction digests.

Solutions:

• SCE

SCE		Final concentration
2M Sorbitol	50 ml	1.0 M
1M Sodium citrate	10 ml	0.1 M
0.25M EDTA, pH 7.0	24 ml	60 mM
sterile ddH2O	16 ml
	-------
	100 ml

• Filter sterilize and store at room temperature.

• Large Scale Prep Lysis Buffer:

• 0.5 M Tris-HCl , pH 9.0

3% Sarkosyl

0.2 M EDTA

References:

Carle, G.F., and M.V.Olson. (1984) "Separation of chromosomal DNA molecules from yeast by orthogonal-field-alteration gel electrophoresis." Nucleic Acids Res. 12:5647-5664.

Burke, D. T., Carle, G. F., and M.V.Olson."Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors." (1987) Science 236:806-812.