Bio-Rad CHEF-DR II System - Manual (Page 17)

3.5 Preparation of Agarose Embedded Yeast DNA
The buffers, enzymes, and agarose found in the following procedure are provided in the
CHEF Yeast Genomic DNA Plug Kit (catalog number 170-3593; see Section 9 for more
information).
1. Inoculate a single colony into 50 to 100 ml YPG broth or appropriate media. Grow with
aeration to an OD
600
of >1.0 at the appropriate temperature for your strain.
2. When the desired OD
600
is reached, centrifuge the cells at 5,000 x g, 10 minutes at
4 °C. Decant the supernatant and resuspend in 10 ml cold 50 mM EDTA, pH 8.
3. Determine the cell concentration by adding 10 µl of cells to 990 µl of water. Place the
yeast suspension on a hemocytometer and count at 400x power. See Section 3.7 for hemo-
cytometer usage.
4. Prepare a 2% CleanCut agarose solution using sterile water and melt using a microwave.
Equilibrate the solution to 50 °C in a water bath.
5. Remove 6 x 10 8 cells for each ml of plugs to be made. Centrifuge in a microfuge for 3 min-
utes if volumes are small, otherwise centrifuge the cells at 5,000 x g, for 10 minutes at
4 °C. Resuspend the cells in one-half the final volume of plugs to be made using Cell
Suspension Buffer (10 mM Tris, pH 7.2, 20 mM NaCl, 50 mM EDTA) and equilibrate
the cell suspension to 50 °C.
6. Just prior to mixing the cells with agarose, add Lyticase to a final concentration of
1 mg/ml for each ml of plugs to be made, to the cell suspension and immediately pro-
ceed with step 7.
NOTE: It is recommended that Lyticase be added immediately prior to imbedding the
cells in agarose. It has been found that certain strains of yeast do not give acceptable
DNA when Lyticase is added after the cells have been imbedded into agarose.
7. Immediately combine the cell suspension with an equal volume of 2% CleanCut agarose
and mix gently but thoroughly. Keeping the cell/agarose mixture at 50 °C, transfer the mix-
ture to plug molds using sterile transfer pipettes (Bio-Rad’s disposable transfer pipettes
catalog 223-9524 are recommended). Allow the agarose to solidify. This step can be
expedited by placing the molds at 4 °C for 10-15 minutes, and it also adds strength to the
agarose for removal from the mold.
8. Using a 50 ml conical centrifuge tube, add 5 ml of lyticase buffer (10 mM Tris, pH 7.2,
50 mM EDTA, 1 mg/ml lyticase) for each one ml of plugs. Push the solidified agarose
plugs, using the snap off tool provided on the plug mold, into the 50 ml centrifuge tube
containing the lyticase buffer. Incubate the plugs 30 minutes to 1 hour at 37 °C without
agitation.
9. Remove the lyticase buffer and rinse the plugs with 25 ml of 1x wash buffer (see step 10
for wash buffer recipe). Add 5 ml of Proteinase K Reaction Buffer (100 mM EDTA, pH
8.0, 0.2% sodium deoxycholate, 1% sodium lauryl sarcosine, 1 mg/ml Proteinase K) for
each ml of agarose plugs. Incubate the plugs overnight at 50 °C without agitation.
NOTE: various cell lines have been incubated up to 4 days in Proteinase K without detri-
mental effects to the quality of DNA’s.
10. Wash the plugs four times in 50 ml of wash buffer (20 mM Tris, pH 8.0, 50 mM EDTA),
30 minutes to 1 hour each at room temperature with gentle agitation. If the plugs are to
be used in subsequent enzymatic reactions, it is advisable to wash the plugs in 1 mM
PMSF during the second or third wash to inactivate any residual Proteinase K activity.
11. Store the plugs at 4 °C. The plugs are stable for 3 months to 1 year.
14