Schematic drawing of DNA agarose discs preparation (adapted from Chung et al. ). Source: H. de Lencastre, ITQB. Portugal)
Pipette 150 μl of the OD620 = 5.0 cell suspensions into a new set of 1.5 ml centrifuge tubes.
Transfer the centrifuge tubes to a 42 °C water bath and incubate for 10 min.
Take the first tube and add 150 μl of the low melting agarose (which has been at 42 °C) and vortex briefly.
Deposit 20 μl droplets onto the parafilm-coated large glass plate (Fig. 1; use 200 μl pipette tips).
Deposit six 20 μl droplets in one row, and in a parallel line deposit a second set of six 20 μl droplets (i.e., a total of 12 droplets for each strain).
Cover the two times six droplets with a microscope slide (Fig. 1).
When all agarose droplets are lined up on the glass tray covered with the microscope slides, transfer the entire glass tray to the freezer compartment of a refrigerator (−20 °C) for 5 min (do not keep longer than 5 min at −20 °C).
Take out the glass tray from the refrigerator and keep it at room temperature for 10 min.
Carefully lift off the microscope slides (the agarose droplets are now ready to be used as “agarose DNA discs” for the rest of the PFGE protocol).
3.5 Lysis and Treatment with Proteinase K
Remove the agarose discs one by one (from the previous step) into 15 ml plastic tubes containing 1 ml of EC lysis solution in each (use disposable sterile loops to transfer the agarose discs into the tubes; all 12 or more agarose discs containing the same DNA go into the same tube) (see Note 7 ).
Incubate the agarose discs in EC lysis solution at 37 °C for 3 h (make sure all discs are submerged in the EC lysis solution).
Decant carefully the EC lysis solution (use sterilized hydrophilic gauze or muslin cloth) leaving the agarose discs in the 15 ml tube.
Remove the last amount of liquid using a 200 μl tip and a pipette.
Add 1 ml of ESP solution to each of the 15 ml tubes containing the agarose discs.
Incubate at 50 °C overnight (17 h) (no agitation/stirring necessary).
Decant the ESP solutions.
Add to each tube 13 ml of TE buffer and wash the discs with gentle agitation for at least 30 min at room temperature (during agitation, the 15 ml tubes are in horizontal position, fixed on a shaker).
Repeat the washing step for 8 times (at least 30 min each) to remove the proteinase K.
After discs have been washed, they can be stored in TE buffer at 4 °C for at least 3–4 months.
3.6 Restriction with SmaI
Transfer 1 DNA agarose disc into a 1.5 ml microtube containing 150 μl of pre-SmaI restriction buffer.
Incubate at room temperature for 30–40 min and remove the buffer with a pipette.
Add 45 μl of SmaI restriction buffer plus 20 units of SmaI enzyme stock per DNA disc (check the equivalent volume in μl) (see Note 3 ).
Incubate at 25 °C overnight.
Add 5 μl of loading buffer (the preparation stays at room temperature while the gel is prepared; if the discs are not immediately used, add 10 μl of ES to the preparation and store the discs at 4 °C for up to 2 days).
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3.7 Preparation of the Gel and PFGE Apparatus
Prepare agarose, running buffer, and sealing agarose (see Subheading 2.1).
Clean with distilled water all parts of the casting tray.
Assemble apparatus, be sure that it is perfectly horizontal, and add comb.
When agarose is ready, pour it in the apparatus slowly to avoid trapping air bubbles (which have to be removed with a pipette tip).
Leave the gel to solidify for at least 30 min at room temperature (if the gel is not going to be used within the first hour, wrap it in Saran wrap and keep at 4 °C).
Rinse the machine three times with 2 L of autoclaved distilled water. Put the frame into the chamber; add 2 L of running buffer, and turn on the machine, the cooling system, and the variable pump, which should be set at 70. Temperature of cooling system is 11.3 °C. Run parameters: initial time of 5 s, final time of 35 s, running time of 23 h, and voltage of 200 V (6 V/cm).
Insert the discs in the gel: take out the agarose disc from the 1.5 ml microtube with a sterile disposable plastic loop, deposit it on the glass slip, and blot off excess liquid with a soft hand paper towel; with the help of the loop, slide the agarose disc into the first well. Make sure the disc is positioned at the bottom and front part of the well (see Note 8 ).
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