8 Pulsed-Field Gel Electrophoresis: Protocols
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• Lysis buffer (155 mM NH 4 Cl, 10 mM KHC03, 0.1 mM EDTA (pH 7.4)) Solutions
• PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HP04, 0.24 g KH2P04 in 11, adjust to
pH 7.4)
• PMSF stock solution (40 mglml in isopropanol)
• Proteinase buffer (1 o/o sodium lauroyl sarcosinate (Sigma), 0.5 M EDTA
(pH 8}}
• TE (10 mM Tris base (pH 8.0}, 1 mM EDTA)
• Distilled water
• Proteinase K
• Falcon tubes
• Neubauer chamber
• Block formers
• Glass plate
• Sterile plastic loops
• Prepare a 2o/o agarose solution in PBS and keep at 45°C.
• Pre-cool ethanol-cleaned block formers on a glass plate sitting on ice.
• Have all solutions handy described above.
Day 1: 2-3 h
Day 3:2 h
Procedure
1. Collect 10 ml of whole blood and add 30 ml oflysis buffer. Place on ice for
at least 20 min until red blood cells lyse.
2. Spin at 2000 rpm for 10 min and decant off clear red supernatant; wash
cells once more in lysis buffer. Then resuspend cells well in PBS.
3. Count the cells inaN eubauer chamber by diluting an aliquot of the single
cell suspension.
4. Cells are resuspended in PBS suchthat 1 million cells are contained in 40
~1 PBS ( 1 million diploid mammalian cells contain roughly 10 ~g of genarnie DNA).
Supplies
ln advance
Time required