Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 8)

Note: Various cell lines have been incubated up to 4 days
in Proteinase K without detrimental effects to the quality
of DNA.
11. 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.
12. Store the plugs at 4 °C. The plugs should be stable for
3 months.
Section 5
Restriction Enzyme
Digestion of Plugs
1. Place one plug per digest in a sterile 1.5 ml microcentrifuge
tube. Wash once for 1 hour in 1 ml 0.1x Wash Buffer
(1:100 dilution of 10x stock Wash Buffer) Use 1 ml 0.1x
Wash Buffer per plug. Decant and resuspend in a sufficient
amount of fresh 0.1x Wash Buffer to cover the plugs. This
last wash reduces the EDTA concentration, allowing faster
buffer equilibration with restriction enzyme buffers.
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7. Just prior to mixing the cells with agarose, add 30 µl of
the Lyticase stock, for each ml of plugs to be made, to the
cell suspension.
Note: It is recommended that Lyticase be added imme-
diately prior to imbedding the cells in agarose. It has been
found that certain strains do not give acceptable DNA
when Lyticase is allowed to diffuse into the agarose plug.
8. 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.
9. Push the solidified agarose plugs into a 50 ml conical
centrifuge tube containing Lyticase solution. Prepare
Lyticase solution by adding 85 µl of Lyticase stock to
2.5 ml of Lyticase Buffer for each 1 ml of plugs. Incubate
the plugs for 2 hours at 37 °C.
Note: For processing a few plugs, use a 2 or 5 ml tube.
10. Remove the lyticase 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.
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