Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 14)

3.4 Preparation of Agarose Embedded Bacterial DNA
The buffers, enzymes, and agarose in the following procedure are provided in the CHEF
Bacterial Genomic DNA Plug Kit (catalog number 170-3592; see Section 9 for information).
1. Inoculate a bacterial culture into 50 ml of LB Broth or appropriate media and grow with
agitation to an O.D.
600
of 0.8–1.0 at the appropriate temperature.
2. When the desired O.D.
600
is reached, add chloramphenicol to a final concentration of
180 µg/ml and continue incubation up to 1 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 suspension on
a hemocytometer and count at 400x power. See Section 3.7 for hemocytometer 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 5 x 10 8 cells for each ml of agarose plugs to be made. Centrifuge for 3 minutes
in a microcentrifuge. If the volume is too large, spin at 10,000 x g for 5 minutes at
4 °C in an appropriate size tube. 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.
Caution: Some bacteria may be sensitive to the concentration of EDTA or the osmotic
strength of cell suspension buffer resulting in premature lysis of the bacteria. This pre-
mature lysis will result in DNA that is unacceptable for PFGE. Bacteria such as
Enterococci require 1 M NaCl in the buffer to prevent osmotic imbalance resulting in
lysis. Pseudomonas is sensitive to EDTA concentration, and dilution of the buffer may be
necessary. Most bacteria require no alteration of the buffer, but as stated in the above
procedure, mixing and imbedding of the bacteria should proceed as quickly as possible.
6. Combine the cell suspension with an equal volume of 2% CleanCut agarose and mix gen-
tly 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 sterile transfer
pipettes (Bio-Rad’s disposable transfer pipettes catalog number 223-9524 are recom-
mended). Allow the agarose to solidify. This step can be expedited by placing the molds
at 4 °C for 10–15 minutes. It also adds strength to the agarose for removal from the mold.
7. Using a 50 ml conical centrifuge tube, add 5 ml of lysozyme buffer (10 mM Tris, pH 7.2,
50 mM NaCl, 0.2% sodium deoxycholate, 0.5% sodium lauryl sarcosine, 1 mg/ml
lysozyme) for each ml of agarose plugs, (e.g. use 25 ml of lysozyme 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 lysozyme buffer. Incubate the
plugs 30 minutes to 1 hour at 37 °C without agitation.
Note: Bacteria such as Staphylococcus aureus and some others are insensitive to lysozyme,
therefore lysostaphin must be substituted for lysozyme buffer. Additionally, adding
lysostaphin to the cell suspension immediately prior to embedding with agarose produces
high quality S. aureus plugs.
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