Bio-Rad CHEF-DR II System - Manual (Page 16)

5. Remove 5 x 10 8 cells for each ml of agarose plugs to be made. Centrifuge for three min-
utes 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 1M 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. 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 num-
ber 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.
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 in place of lysozyme buffer.
Additionally, adding lysostaphin to the cell suspension immediately prior to imbedding
with agarose produces high quality S. aureus plugs. See Section 3.5, step 6 for a more
detailed description of the procedure.
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 DNAs.
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 one 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 3.7 for more information
on restriction digestion of plugs.
13