Bio-Rad CHEF Genomic DNA Plug Kits User Manual

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7. Wash the plugs four times in 1x Wash Buffer, 1 hour each

at room temperature with gentle agitation (for each plug,
use 1 ml of 1x Wash Buffer). Prepare the Wash Buffer by
diluting the 10x stock (1:10) with sterile ddH

O. If the

plugs are to be used in subsequent enzyme reactions, it is
advisable to wash the plugs in 1 mM PMSF during the
second or third wash to inactivate residual Proteinase K.
See Appendix for PMSF stock solution.

Note: For washing a few plugs, use a 2 or 5 ml sterile tube.

8. Store the plugs at 4 °C in 1x Wash Buffer. The plugs

should be stable for 3 months.

Section 3
Preparation of Agarose Embedded
Bacterial DNA

Reagents and Equipment Needed

Sterile transfer pipettes
50 °C water bath
Grams Crystal Violet (Difco)
Microscope
PMSF stock solution see Appendix
Hemocytometer see Appendix

2, 5, 50 ml sterile plastic tubes

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. See appendix
for LB Broth.

3. Calculate the amount of Cell Suspension Buffer

and CleanCut agarose necessary (see Section 9.2). For a
final concentration of 0.75% agarose use 0.63 ml of Cell
Suspension Buffer per ml of agarose plugs. Use 0.37 ml
of 2% CleanCut agarose per ml of agarose plugs.

4. Centrifuge the cell suspension at 1,000 x g for 5 minutes

at 4 °C. Resuspend the cells in the volume of Cell
Suspension Buffer calculated above and equilibrate the
cell suspension to 50 °C.

5. 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.

6. Using a 50 ml conical centrifuge tube, add 100 µ l of

Proteinase K stock to 2.5 ml of Proteinase K Reaction
Buffer for each ml of agarose plugs. Push the solidified
agarose plugs 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 detrimental effects to the quality
of DNA.

Note: For processing a few plugs, use a 2 or 5 ml tube.

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