When preparing high molecular weight DNA for Optical Mapping, it is important to protect the molecules from mechanical breakage and nucleolytic degradation. The same general methods for preparing large DNA for pulsed field gel electrophoresis (PFGE) are currently used for Optical Mapping. In general, cells are embedded in low melting temperature agarose, treated to form spheroplasts and digested with proteinase K. Cellular debris is removed in a series of washes leaving just the DNA trapped in the agarose. Gentle warming and treatment with agarase releases the DNA for Optical Mapping analysis. This process typically yields DNA with an average molecule length between 250 and 500 kb, though megabase long molecules are often observed.

1.  Obtain a fresh liquid culture

The highest quality DNA is often obtained from fresh liquid cultures that have reached the mid- or late log phase of growth. However, for some fragile organisms it may be necessary to harvest the culture in early log phase.

Once the culture has reached the desired growth phase, collect the cells by centrifugation and discard the supernatant. Wash the cells by resuspending the cell pellet in 1/5 the original culture volume with cell suspension solution (200 mM NaCl, 10 mM Tris, pH 7.2, 100 mM EDTA) and centrifuging again.

2.  Concentrate the culture

After centrifugation, discard the supernatant and resuspend the cell pellet in the appropriate volume of cell suspension solution to yield 5 x 109 cells / ml. For genomes smaller than 2 - 3 Mb, a 1010 cell concentration is desired.

For well characterized strains, the cell concentration may be easily estimated by measuring the O.D. 600 of the culture at the time of harvest. In other cases, it may be necessary to determine the cell count with other methods. Counting the cells with a hemocytometer will produce the most reliable estimate.

3.  Embed the cells in agarose

Add an equal volume of 1% low melting temperature agarose to the cell suspension for a final 0.5% agarose solution. We recommend using SeaPlaque GTG agarose (Cambrex Catalog #50111). The agarose should be pre-cooled to 50°C. Mix the solution well by gentle pipetting and dispense the mixture to agarose plug molds (BIORAD Catalog # 170-3713). Each plug holds approximately 100 µl. Place the plugs at 4°C for 30 minutes.

4.  Generate spheroplasts

Bacterial cell walls are quite variable and the exact spheroplasting treatment depends on the organism. For some organisms, the spheroplasting step can be skipped. The procedure outlined here is intended for organisms that are sensitive to lysozyme.

Transfer the agarose plugs to a 50 ml conical screw-cap tube containing bacterial lysis solution (10 mM Tris pH 7.5; 50 mM NaCl; 100 mM EDTA; 0.2% Na deoxycholate; 0.5% sarcosyl, Na salt; 1 mg / ml lysozyme). For 4 - 8 plugs, use 15 ml of solution. Incubate the plugs at 37°C for 2 - 16 h. The incubation time depends on the organism's sensitivity to lysozyme.

5.  Digest with Proteinase-K

Transfer the plugs to a new 50 ml tube containing 15 ml of digestion solution (0.5 M EDTA, pH 9.5; 1% sarcosyl; 2 mg/ml Proteinase-K). Incubate at 50°C for 48 h, replacing with fresh digestion solution after 24 h.

6.  Rinse the plugs

The plugs should be washed three times in 0.5 M EDTA, pH 9.5. For each wash, transfer the plugs to a fresh 50 ml conical screw-cap tube containing 40 ml of EDTA.  Incubate the tube for 1 hour at room temperatue, rocking slowly.  After the last wash, store the plugs in a microfuge tube (no more that 2 plugs/tube) containing 0.5 M EDTA.  Each tube should be filled completely with 0.5 M EDTA.  Make sure that each tube is clearly labeled, including the strain ID and tube number corresponding to the Sample Submission Form.  Make sure the lids of the tubes are secured by wrapping in parafilm or by taking other precautions to prevent spills. Store the plugs at 4°C until shipment.  Do not allow the plugs to freeze.

7.  Shipment to OpGen

Complete the Sample Submission Form and include a copy with the sample shipment.  For each strain, ship 4 plugs by overnight carrier, on ice.