Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 13)

3.2 Liquid Samples
High molecular weight DNA can be prepared by standard procedures. DNA fragments of
up to several hundred kilobases do not require preparation in agarose blocks, and can be added
to the wells in liquid form. When working with DNA in the range of 50–200 kb, it may be nec-
essary to use pipette tips with large openings. When running only liquid samples, the best
resolution and sharpness of bands is achieved using a thin well comb (0.75 mm).
3.3 Preparation of Agarose Embedded Mammalian DNA
The buffers, enzymes, and agarose in the following procedure are provided in the CHEF
Mammalian Genomic DNA Plug Kit (catalog number 170-3591; see Section 9 for information).
1. Prepare a cell suspension in isotonic saline or tissue culture medium without fetal bovine
serum. Count the cells and remove 5 x 10 7 cells for each ml of agarose plugs to be made
and place on ice. See Section 3.7 for hemocytometer use. The 50 well plug mold makes
5 ml of agarose plugs. We recommend making slightly more than 5 ml if all fifty wells
are to be used.
2. Prepare a 2% low melt agarose (2% CleanCut ™ agarose is recommended, catalog num-
ber 170-3594) solution in sterile water and melt using a microwave. Equilibrate the solu-
tion to 50 °C in a water bath.
3. Centrifuge the cell suspension at 1,000 x g for 5 minutes at 4 °C. Resuspend the cells in
one-half the final volume of plugs to 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.
4. Combine the cell suspension with an equal volume of 2% CleanCut agarose and mix gen-
tly but thoroughly. This results in a final concentration of 1% agarose. 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 number 223-9524, are recom-
mended). Allow the agarose to solidify. This step can be expedited by placing the molds
at 4 °C for 10–15 minutes. This also adds strength to the agarose for removal from the
mold.
5. Using a 50 ml conical centrifuge tube, 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 (e.g. use 25 ml of Proteinase K
Reaction 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
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 detri-
mental effects to the quality of DNA.
6. 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.
7. Store the plugs at 4 °C. The plugs are stable for 3 months to 1 year.
8. 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 3.6 for information on
restriction digestion of plugs.
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