Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 5)

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
2
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.
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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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