Charles ANDRE and Virginia WALBOT
Department of Biology, Stanford University, Stanford Ca. 94305, USA
1. Isolation of mitochondria from seedlings. Mitochondria are isolated from seedlings as in ref. 1. Resuspend final mitochondrial pellet (from step "f") in phosphate buffered saline (PBS- 130 MM NaCl, 10 mM Na2HP04, 3 mM KCl, 2 mM KH2PO4, pH 7.4) (1 ml per 100 g seedlings). Proceed to step 5. For isolation of mitochondria from tissue culture cells, see below.
2. Making protoplasts. Pellet tissue culture cells 5', 1,000 rpm. Resuspend (1:1 of original culture volume) in protoplast isolation media (PIM- .735 g/l CaCl 2 . 2H20, 100.2 g/l manitol, .976 g/l MES, pH 5.8). Centrifuge as above, resuspend in an equal volume PIM with enzymes (cellulase (Onofuka) 1.8 g/100 ml, macerozyme R-10 (Honsha) 1g/100 ml, Bovine Serum Albumin (Sigma) 0.5 g/100 ml, B mercaptoethanol (Sigma), 15μl/100 ml.) Let cells digest for 2-3 hours at 25°C with gentle shaking (40 rpm). Check protoplasts by taking 10μl and observing at 40X under a microscope (protoplasts should be perfectly round and not clumped together.)
3. Lysing protoplasts. Protoplasts are gently broken by squeezing through a nylon mesh. Resuspend protoplasts in cold PBS (about 10 ml/100 ml starting culture) draw into a 12 ml syringe to a millipore filter unit fitted with a piece of 100 mm mesh, and squeeze protoplasts through (into a 15 ml falcon 2059 tube on ice). This will break up any remaining clumps of cells/protoplasts. Filter again through 10μm mesh to break protoplasts. Check lysis by looking at a sample under microscope (few protoplasts and lots of cell debris should be seen).
4. Differential centrifugation. Spin lysate at 3,500 rpm (1,500 X g) in Sorvall RC5-B at 4°C for 10 minutes to remove large debris and nuclei. Pour supernatant into a cold 2059 tube and spin again at 9,000rpm (9,50OXg) to pellet the mitochondria. Pour off supernatant and resuspend the resulting pellet in cold PBS with a fine brush. Resuspend in 0.5 ml PBS for every 100 ml starting culture.
5. Making the plug. Prepare agarose ahead of time (1.2% low-melt sea-plaque or InCert agarose (FMC) in 1xPBS) and place at 47°C. Add equal volumes of mitochondrial suspension and agarose, mix quickly, and immediately pipette into a Bio-rad plug mold (each well of the mold holds 200μl). Put plug mold on ice and let plugs solidify for 5-10 minutes.
6. Lysing plugs. Put plugs in a tube containing 1% SDS, 0.1 M EDTA and 1 mg/ml proteinase K. Shake at 37°C overnight. (Plugs should become clear).
7. Processing plugs. Remove lysis solution and resuspend plugs in TE100 (Tris- HCl 10 mM, EDTA 100 mM, pH 7.5). Inactivate Proteinase K by adding phenylmethylsulfonylfluoride (PMSF) to a final concentration of 1 mM. Place at 37°C for 1-2 hours. Remove PMSF by several washes with TE100. Plugs may be stored in this solution at 4°C for a year or more.
8. Restriction enzyme digestion. The plugs are soaked in 1 ml of the appropriate restriction buffer for 1 hour on ice, then moved to a tube containing 100-200μl of restriction buffer plus enzyme. Digestion is allowed to proceed overnight.
9. Pulsed-field gels. The digested plugs are soaked in 0.5 x TBE (TBE-45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.0) for 1 hour, then placed into the wells of a 1% agarose gel made with 0.5 x TBE. The wells are sealed with 0.6% Sea-plaque agarose (FMC) and allowed to solidify. Gels are run in a CHEF apparatus (Bio-Rad CHEF-DR II), using a ramped pulse time of 1- 15 seconds at 175 volts for 20 hours. These conditions resolve DNA in the range of 5-250 kb.
Mulligan, R. M., Dement, E., Andre, C., and V. Walbot, 1988. isolation of RNA and DNA from rice seedling mitochondria. RGN 5: 151-154.