TITLE: Visualization of Yeast Chromosomes Using Clamped Homogeneous Electric Field (CHEF) Electrophoresis
AUTHORS: Lois L. Hoyer

[DESCRIPTION]
I inherited this protocol while working as a postdoctoral researcher in the laboratory of Dr. Stewart Scherer in the 1990s. Literature that influenced protocol development is listed in the References section. 

This protocol describes how to isolate chromosome-sized DNA from yeast cells. The yeast cells are encased in agarose, then the cell is digested away. The result is unsheared chromosomes in an agarose plug that can be loaded onto electrophoresis gels or subjected to additional analysis such as digestion with restriction enzymes.

The protocol details two sets of electrophoresis conditions for separating the chromosomes using a BioRad CHEF-DR III variable angle electrophoresis system. Because the protocol was developed for work with Candida albicans, the conditions are most useful for chromosomes in the size range of approximately 0.5 to 5 Mb. The "short protocol" (Condition 1) separates the smaller C. albicans chromosomes (approximately 1 Mb) without running them off the bottom of the gel. The "long protocol" (Condition 2) separates the larger chromosomes (approximately 3 Mb) with approximately 0.1 Mb resolution but risks running smaller DNA molecules off the gel. We typically run samples using both electrophoresis protocols and combine the resulting information to understand the karyotype.

[STEPS]
SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
1. Grow the yeast strain on an agar plate, streaking for isolated colonies. Plates can be stored at 4 °C until needed.


 


Select a well-isolated, representative colony. Inoculate it into the liquid growth medium of your choice. Cultures can be incubated to saturation or harvested at an earlier time point, as long as a sufficient number of cells are available (see below).

 

 


SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
3. Count the yeast cells using a hemacytometer.
For "1x" plugs, use 2 x 108 cells total for a single recipe; for "2x" plugs, use 4 x 108 cells total.

 

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
4. Remove the culture volume required for the desired number of cells. Collect the cells by centrifugation. Wash the cells twice in  50 millimolar (mM) EDTA, pH 8.0.
SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
5. Resuspend cells in 200 µL 50 millimolar (mM) EDTA, pH 8.0.

Since the presence of the yeast cells increases the overall volume in the microfuge tube, measure the true volume using a Pipetteman. Set the Pipetteman for a larger volume than expected, pull up the entire volume into the pipette tip and carefully dial the Pipetteman back until the liquid is just at the end of the pipette tip. Read the Pipetteman volume and make a note of it for Step 7 (below). 

 

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
6. Prepare the following Zymolyase-containing solution: 
      Final solution  Stock  Vol. needed for 250 ul    50% glycerol  50% glycerol  250 pl    2.5 mg/ml yeast lytic enzyme  Powder  0.625 mg    10 mM sodium phosphate, pH 7.5  1 M  2.5 pl  
 Mix well with a Pipetteman to ensure that the Zymolyase powder is dissolved.

Add 15 µL of Zymolyase-containing solution to the cell suspension and mix by pipetting.
SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
2. Prepare 1.5% Low-Melting-Point (LMP) agarose in 125 millimolar (mM) EDTA, pH 8.0 .

Recipe:
Mix 10 mL of 0.5 Molarity (m) EDTA pH 8.0 with 30 mL purified water. 

Add 0.6 g Low-Melting-Point agarose. Heat in a microwave or on a heating plate to dissolve the agarose.

Since water will evaporate during this process, add purified water back to adjust the volume of the solution to 40 mL . Since adding water decreases the temperature of the agarose solution, it may be helpful to have a bottle of purified water pre-warmed for this purpose.

The final agarose solution should be cooled to 37 °C prior to use in Step 7 below.

 

 

 


 

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
7. Note the volume for each sample you are preparing. Total volume = (EDTA + cell volume measured in Step 5) + 15 µL Zymolyase-containing solution from Step 6.

Make a 1:1 mix of the cell preparation with the LMP agarose. In other words, add an equal amount of LMP agarose (from Step 2) to the tube containing the cells. Mix well and immediately distribute into plug molds.

 

Allow filled molds to cool at 4 °C for at least 30 min.
SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
8. Remove the solidified agarose plugs from the molds.

When using the thinner mold, final plugs are larger than the electrophoresis gel well. The plugs can be cut into equal thirds using a razor blade. Working on a chilled glass plate keeps the plugs solid during the process of cutting them.

Plugs will be incubated overnight at 30 °C for the Zymolyase digestion to occur. Plugs can be placed into tubes, or into the wells of a 6-well tissue culture plate. It is easier to handle the plugs if they are placed into the wells of a 6-well tissue culture plate.

 

Plugs will be incubated in a Tris/EDTA solution that is made by mixing: 
0.2 ml 1 M Tris-HCl, pH 8.0
20 ml 0.5 M EDTA, pH 8.0

Add several ml of the solution to each well in the tissue culture plate. Distribute the plugs into the wells as needed for your experiment. Use a flat spatula (e.g. like those used for weighing chemicals) to lift each plug and release it into the tissue-culture-plate well. Make sure the plugs are covered with the buffer and add a little extra to accommodate moderate evaporation during incubation.

Incubate at 30 °C overnight without agitation.
SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
9. Remove the tissue-culture plate from the incubator and pipette off the incubation buffer. Wash the plugs three times in 0.5 Molarity (M) EDTA pH 9.5 (15-30 min per wash). Agitate the plate gently during washing.

 

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
10. The plugs will next be subjected to Proteinase K digestion, which is more efficient at a higher temperature. The plugs should be placed into a round-bottom tube with a cap that can limit evaporation. We use 14-ml round-bottom tubes like Nunc catalog number 150268, but many other choices would work well.

Proteinase K Digestion Solution Recipe:
Since Proteinase K is expensive, prepare only the amount of this solution that is needed for the number of plugs being prepared.

      Final solution  Stock  Vol. needed for 5 ml     2.5 mg/mI Proteinase K  Powder  12.5 mg    1% Sarcosyl  10%  0.5 ml    0.5 M EDTA pH 9.5  0.5 M  5 ml    10 mM Tris-HCI, pH 9.5  1 M  50 µl  
Mix the proteinase K digestion solution well and distribute a small amount into each tube. Carefully place the plugs into the tube. All plugs from the same species/strain can be placed in a single tube. Add more digestion solution to ensure that the plugs are covered completely. Place the cap on the tube tightly.

Incubate 30 min at 50 °C.

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
11. Carefully pour the plugs from the tube into a well on a 6-well tissue culture plate. Wash plugs three times with 50 millimolar (mM) EDTA pH 8.0 working similarly to Step 9.

SECTION: How to Make Agarose-Embedded Yeast Chromsomes (Chromosome Plugs)
12. Store plugs at 4 °C in 50 millimolar (mM) EDTA pH 8.0. Plugs may be stored in the tissue culture plate, but evaporation of buffer occurs over time. Place them in a capped tube or check the plates at routine intervals to ensure that the plugs do not dry out.
 


SECTION: Separating Yeast Chromosomes on a Pulsed-Field Electrophoresis Gel
14. Prepare the buffer and agarose gel.

Separation of chromosome-sized DNA is best accomplished using Pulsed Field Certified Agarose (e.g. Bio-Rad catalog number 162-0137). 

Both sets of gel running conditions described below use 1x Tris-Acetate-EDTA (TAE) buffer. TAE buffer is made at a 50x concentration and diluted to 1x just before use.

50x TAE Recipe
 
Prepare the gel by dissolving the correct amount of Pulsed Field Certified Agarose in 1x TAE buffer. For example, the protocol called Condition 1 uses a 0.8% agarose gel. Preparing 150 ml of agarose will fill the Bio-Rad gel tray with a little extra left over. The leftover agarose is used to seal the gel wells after the plugs are loaded.

To prepare 150 ml of 0.8% agarose in 1x TAE buffer, weigh 1.2 g Pulsed Field Certified Agarose and dissolve it in 150 ml 1x TAE buffer. Heat in a microwave or on a heating plate. A stir bar may be used on the heating plate. Cool the agarose to less than 60 °C before pouring the gel to avoid warping the gel tray.

Pulsed field gels work best when poured on a level surface. Place the gel tray onto a leveling platform and use a bubble level to adjust. Pour the cooled agarose into the gel tray and place the comb so the teeth are closest to the frame of the gel tray. Placing the comb in the opposite orientation shortens the running length of the gel. 

Allow the gel to solidify at room temperature.
SECTION: Separating Yeast Chromosomes on a Pulsed-Field Electrophoresis Gel
13. The CHEF system in my laboratory includes the variable-speed pump for buffer recirculation and the cooling module. 

Two sets of running conditions are presented below. These conditions are for use on a BioRad DR-III device. Modifications will be needed for other instruments. Both sets of conditions use 1x TAE buffer. 

The running conditions were developed for use with C. albicans chromosomes. The diploid C. albicans has eight chromosome pairs that range in size from chromosome 1 (3.2 Mb) to chromosome 7 (0.95 Mb). Chromosome R (that encodes variable rDNA copy numbers) is large, migrating in the size range of chromosome 1.

Condition 1 will resolve all but chromosomes 1 and R, leaving room on the gel for chromosome fragments smaller than chromosome 7.

Condition 2 will separate chromosomes 1 and R but runs chromosome 7 to near the bottom of the gel.
SECTION: Separating Yeast Chromosomes on a Pulsed-Field Electrophoresis Gel
16. Load your plugs into the gel wells. This step takes a little practice. You will likely break some plugs before you can load them into the gel cleanly and without bubbles. It helps to add 100 µL of 1x TAE (running buffer) to the gel well, which fills it approximately half-way. Plugs tend to slide into the well more easily with the buffer present. Filling the well completely with 1x TAE tends to repel the plug from entering the well.

Once plugs are loaded, use the flat spatula to ensure that the plug is "flush" with the leading edge of the gel well. In other words, manipulate the plug so that its face meets the leading edge of the gel well without air bubbles or gaps. This step also takes practice. You can also use a Hamilton syringe to remove air bubbles from the well.

Once the plugs are positioned correctly, use the remaining Pulsed Field Certified Agarose/1x TAE solution to seal the plugs into the wells. This step prevents the plugs from floating away during the electrophoresis step.
SECTION: Separating Yeast Chromosomes on a Pulsed-Field Electrophoresis Gel
17. Place your gel into the gel box. Allow the gel to equilibrate to the temperature of the buffer before starting the electrophoresis protocol.

Set up the CHEF power supply using parameters for either Condition 1 or Condition 2:

Condition 1: 0.8% agarose gel run at 14 °C at 3 V/cm with a 106-degree angle. Running time is 3000 min with a 120-480 sec linear ramp.

Condition 2: 0.6% agarose gel run at 12 °C at 2.5 V/cm with a 120-degree angle. Running time is 1440 min with a 120-300 sec linear ramp followed by 2760 min with a 420-900 sec linear ramp.

Examine the depth of the buffer in the gel box. Ideally, the buffer should just cover the top surface of the gel. Drain excess buffer from the box.

Start the power supply and ensure that the gel is running. If you drained too much buffer in the previous step, for example, there may not be bubbles coming from the electrodes in the box. Note the milliamps at the start of the run. We typically see values of 150 mA at the start of a run; the value increases as the run progresses. If the reading is too high, try draining more buffer from the gel box. Bio-Rad literature discusses this point in detail, suggesting that the mA reading is also affected by water quality or buffer preparation problems.

Monitor the run as it progresses. Ensure that there are no leaks, that the buffer level in the gel box is still acceptable, and that the chiller unit is maintaining the desired temperature.

When the run is completed, drain the buffer from the gel box and rinse the box/tubing with water in preparation for storage or future use.
SECTION: Separating Yeast Chromosomes on a Pulsed-Field Electrophoresis Gel
15. Add 1x TAE to the gel box. Start the recirculation pump and the cooling module. Select either 14 °C (Condition 1) or 12 °C (Condition 2) depending on the protocol you would like to run. It takes a little time for the buffer to reach the selected temperature so set up the gel box before loading the gel.