(a) Prepare a "slot well" by joining 4-6 wells together. If necessary, the slot-well can be extended anteriorly to provide more space for the plug pieces. Seal the bottom of the slot-well with a thin layer of melted agarose.

(b) Place plug pieces into the slot-well. Load the PFGE lambda ladder in wells flanking the slot well. Seal all wells with melted agarose.

(c) Divide the gel into three pieces as shown. The two flanking pieces should each contain a small part of the slot well as well as a DNA ladder. Stain the flanking pieces with ethidium bromide. Do not stain the center piece (the gel piece containing most of the genomic DNA).

(d) Align the stained flanking gel pieces and examine on a UV light box. If the digestion worked as planned, most of the genomic DNA should lie between 100 and 350 kb. Using a scalpel, make incisions in the flanking pieces marking the 100 kb and 350 kb borders (pink and green lines, respectively).

(e) Reconstruct the gel by placing the unstained center pieces between the two flanking gel pieces. Extend the incisions at 125 kb and 350 kb on the flanking pieces into the center gel piece (green and pink lines).

(f) Cut the center gel piece transversely to connect the two incisions marking 100 kb (pink line) and the two incisions at 350 kb (green line). Stain the resulting upper and lower pieces. The center block will be used in the second size selection.

(g) Cut the center gel block (containing unstained DNA between 100 and 350 kb) transversely at two spots to produce three agarose blocks of roughly equal size. These blocks (from bottom to top) are referred to as x, y, and z, respectively.