Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 15)

8. Remove the lysozyme buffer and rinse the plugs with 25 ml of 1x wash buffer (see step
9 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.
9. 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.
10. Store the plugs at 4 °C. The plugs are stable for 3 months to 1 year.
11. Maintain the plugs in 1x Wash Buffer for long term storage. However, for subsequent
restriction digestion, the EDTA concentration must be lowered. Wash the plugs to be
restricted for 30 minutes in 0.1x wash buffer or TE. See Section 3.6 for more information
on restriction digestion of plugs.
3.5 Preparation of Agarose Embedded Yeast DNA
The buffers, enzymes, and agarose 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 O.D.
600
of >1.0 at the appropriate temperature for your strain.
2. When the desired O.D.
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 use.
4. Prepare a 2% low melt agarose (2% CleanCut agarose is recommended, catalog number
170-3594) solution using sterile water and melt using a microwave. Equilibrate the solu-
tion 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 proceed with step 7.
Note: Add Lyticase immediately prior to embedding the cells in agarose. Certain strains
of yeast do not give acceptable DNA when Lyticase is added after the cells have been
embedded into agarose.
7. Immediately combine the cell suspension with an equal volume of 2% CleanCut agarose
and mix gently but thoroughly. This results in a final concentration of 1% agarose.
Keeping the cell/agarose mixture at 50 °C, transfer the mixture to plug molds using ster-
ile transfer pipettes (Bio-Rad’s disposable transfer pipettes catalog number 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.
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