Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 6)

of Cell Suspension Buffer calculated above and equilibrate
the cell suspension to 50 °C.
7. Combine the calculated volume of 2% CleanCut agarose
with the cell suspension and mix gently, 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, which also adds strength to the agarose
for removal from the mold.
8. Push the solidified agarose plugs into a 50 ml conical
centrifuge tube containing lysozyme solution. Prepare
lysozyme solution by adding 100 µl of Lysozyme stock
to 2.5 ml of Lysozyme Buffer for each 1 ml of agarose
plugs. Incubate the plugs for 2 hours at 37 °C.
Note: For processing a few plugs, use a 2 or 5 ml tube.
9. Remove the lysozyme solution and rinse the plugs with
sterile water. Add 2.5 ml of Proteinase K Reaction Buffer
for each ml of agarose plugs, followed by 100 µ l of
Proteinase K stock. Incubate the plugs overnight at 50 °C
without agitation.
Note: Various cell lines have been incubated up to 4 days
in Proteinase K without detrimental effects to the quality
of DNA.
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2. When the desired O.D.
600
is reached, add chloramphenicol
to a final concentration of 180 µ g/ml and continue
incubation up to 1 hour while performing step 3.
Note: Chloramphenicol is used to synchronize ongoing
rounds of chromosomal replication 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;
therefore proceed as quickly as possible with step 3.
3. Make a twenty-fold dilution of the above bacterial
suspension using 1 ml bacteria, 1 ml Gram Crystal
Violet, and 18 ml saline or PBS. Place a small amount of
the bacterial suspension on a hemocytometer and count at
400x power. See Appendix on hemocytometer usage.
4. Melt the 2% CleanCut agarose solution using a
microwave or hot water bath and equilibrate the solution
to 50 °C in a water bath.
5. Calculate the amount of Cell Suspension Buffer and
CleanCut agarose necessary (see Section 9.2). For a final
concentration of 1% agarose use 0.5 ml of Cell Suspension
Buffer per ml of agarose plugs (use 100 µ l/plug for
disposable mold or 300 µl/plug for reusable mold). Use
0.5 ml of 2% CleanCut agarose per ml of agarose plugs.
6. 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 min at 4 °C in
an appropriate size tube. Resuspend the cells in the volume
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