Bio-Rad CHEF-DR II System - Manual (Page 15)

4. Combine the cell suspension with an equal volume of 2% CleanCut agarose and mix gen-
tly but thoroughly. Keeping the cell/agarose mixture at 50 °C, transfer the mixture 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.
6. Using a 50 ml conical centrifuge tube, 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 (e.g. use 25 ml of Proteinase K
Reaction Buffer for 5 ml of agarose plugs). Push the solidified agarose plugs, using the
snap off tool provided on the plug mold, into the 50 ml centrifuge tube containing the
Proteinase K solution. 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 DNAs.
7. 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.
8. Store the plugs at 4 °C. The plugs are stable for 3 months to one year.
9. 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 3.7 for more information
on restriction digestion of plugs.
3.4 Preparation of Agarose Embedded Bacterial DNA
The buffers, enzymes, and agarose found in the following procedure are provided in the
CHEF Bacterial Genomic DNA Plug Kit (catalog number 170-3592; see Section 9 for more
information).
1. Inoculate a bacterial culture into 50 ml of LB Broth or appropriate media and grow with
agitation to an OD
600
of 0.8 - 1.0 at the appropriate temperature.
2. When the desired OD
600
is reached, add chloramphenicol to a final concentration of
180 µg/ml and continue incubation up to one hour while performing step 3.
Note: Chloramphenicol is used to synchronize ongoing rounds of chromosomal replica-
tion and inhibit further rounds of replication. This step is optional, but regions near the
replication terminus might be under represented. In addition, chloramphenicol will alter
the morphology of the cells over time causing the appearance of a mixed culture, there-
fore proceed as quickly as possible with step 3.
3. Make a twenty-fold dilution of the above bacterial suspension using 10 µl bacteria, 20 µl
Gram Crystal Violet, and 170 µl saline or PBS. Place a small amount of the bacterial sus-
pension on a hemocytometer and count at 400x power. See Section 3.7 for hemocytometer
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.
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