DNA Preparation
E. coli chromosomal DNA is prepared following the method of Heath et al. ( J. Bacteriol., 174, 1992). Cells are embedded in agarose, then treated with deter gents and enzymes which remove the cell wall, proteins and other cellular material. The purified chromosomal DNA remains embedded in the agarose plug.
Preparation of cell plugs
1-Grow an overnight culture of E. coli MG1655 in L broth. The OD600 of an overnight culture of this strain should be approximately 1.7.
2-Make a 1.6 solution of InCert (FMC) agarose in water. Dissolve the agarose by boiling, then cool in a 50 °C water bath.
3-Pellet cells by spinning 15 min at 5,000 rpm in the Sorvall centrifuge (SA600 rotor).
4-Resuspend cells in an equal volume of PIV buffer.
PIV buffer:
1 M NaCl
10 mM Tris-HCl (pH 7.6)
5-Spin cells as in step 3.
6-Resuspend cells in 1/2 volume PIV buffer.
7-Warm cells to 37 °C.
8-Mix the cells with an equal volume of molten (50 °C) agarose and pipet into the wells of a 24-well microtiter plate. Add 1/2 ml of the cells/agarose mixture to each well.
9-Place plate at 4 °C to allow the agarose to harden.
Lysozyme/RNase digestion
1-Add 1 ml per well EC lysis solution without lysozyme and RNase.
EC lysis solution:
6 mM tris-HCl (pH 7.6)
1 M NaCl
100 mM EDTA
0.5 Brij-58
0.2 deoxycholate
0.5 Sarkosyl
1 mg/ml lysozyme
20 μg/ml RNase
2-Shake gently at room temperature approximately 15 min.
3-Aspirate off the EC lysis solution (be careful not to suck up the agarose plug!).
4-Add 1 ml per well EC lysis solution with lysozyme and RNase.
5-Seal plate inside a heat-sealable bag.
6-Place bag (submerged) inside a shaking water bath. You will have to hold the bag down with lead donuts or other weights. Shake gently overnight at 37 °C .
Proteinase K digestion
1-Aspirate off the EC lysis solution.
2-Add 1 ml per well ESP buffer without proteinase K.
ESP buffer:
0.5 M EDTA (pH 9.0 to 9.5)
1 Sarkosyl
50 μg/ml proteinase K
3-Shake gently at room temperature approximately 15 min.
4-Aspirate off ESP buffer.
5-Add 1.5 ml per well ESP buffer with proteinase K.
6-Seal plate inside a heat-sealable bag.
7-Place inside a shaking water bath as above, and shake gently overnight at 50 °C.
TE washes
1-Aspirate off the ESP buffer.
2-Rinse each well with 1 ml sterile TE buffer (pH 7.5). Aspirate off.
TE (pH 7.5):
10 mM Tris-HCl (pH 7.5)
1 mM EDTA
3-Add 1.5 ml per well sterile TE.
4-Shake at room temperature 30 min.
5-Aspirate off the TE.
6-Repeat steps 3-5 for a total of 6 washes.
7-After removing the last wash, add 1 ml sterile TE to each well and store plugs at 4 °C.
Restriction Digests
The DNA is digested while still embedded in agarose.
1-With a sterile spatula or pair of tweezers, carefully remove a DNA plug from its well and place it on a sheet of parafilm.
2-With a sterile razor blade, slice each plug in half, then slice each half-plug in half again, lengthwise.
3-Place two slices (which together would make up one half-plug) inside a sterile Eppendorf tube containing 900 μl sterile 10 mM Tris-HCl (pH 7.5). The pieces should fit into the bottom of the tube.
4-Place the tube in a rack and shake gently at room temperature approximately 45 min.
5-Remove the TE (be careful not to damage the gel slices) and add 900 μl of the appropriate restriction enzyme buffer. Shake gently at room temperature for approximately 1 hr.
6-Repeat step 5.
7-Remove buffer. Add 200 μl fresh restriction enzyme buffer and enzyme. Use approximately 30 to 60 units of enzyme per reaction.
8-Place tubes in 37 °C incubator overnight.
9-The next day, stop the reaction by adding 10 μl of 0.5 M EDTA to each tube. Store reactions at 4 °C.
Electrophoresis
Pulsed field gel electrophoresis is run using the BioRad CHEF MAPPER TM system. See the instruction manual for complete information on set-up and use of the system.
Gel pouring
1-Prepare a solution of 1 SeaKem GTG agarose (FMC) in
0.5x TBE. Do not add ethidium bromide to the gel solution.
2-Pour the gel according the instructions in the BioRad manual. Use 100 ml agar ose for the small gel tray.
3-Chill gel at 4 °C before loading and running.
Sample loading
Molten agarose samples are loaded as follows:
1-Pre-warm Pasteur pipets by placing several inside a test tube, capping the tube, and placing it in a 70 °C water bath.
2-Re-melt (if necessary) the 1 agarose solution that was used to pour the gel and equilibrate it to 70 °C in the water bath.
3-Remove a gel slice from the restriction digest buffer and rinse with water. Place gel slice inside a clean Eppendorf tube.
4-To melt the gel slice, place tube in the 70 °C water bath, leaving it in just long enough to melt the gel (2 or 3 minutes should be enough time to melt it). Flick the tube often to mix the contents and to ensure the entire sample is melted.
5-As soon as the sample is melted, load it on the gel. Use a
pre-warmed Pasteur pipet to load the sample and work quickly, being careful not to let any air bubbles get entrapped in the wells. Load samples to just flush with the top of the gel.