Mitochondria is separated by differential centrifugation from
the bulk of nuclei, plastids, and cellular debris which differ
in particle size or density. Mitochondria are then further separated
from the remaining plastids and nuclear debris by gradient centrifugation.
Homogenization media for mitochondrial isolation contain an osmoticum,
EDTA, bovine serum albumin (BSA), 2-mercaptoethanol, and polyvinylpyrrolidone
(PVP) which binds phenolics.
Suspension culture is a useful source of nongreen tissue in small-seeded
species lacking fleshy fruit, large storage organs, or convenient
nongreen floral tissue. This strategy should be considered for
species in which axenic vegetative tissue is not easily obtained,
or if green tissues do not yield mtDNA.
Yield of mtDNA; 50-100 ug/ 100 g of packed suspension culture
cells.
MATERIALS AND SOLUTIONS
Bead-Beater
This device breaks cells by agitating them with glass beads in
a small plastic vessel, and is simpler and less expensive than
a French press.
Suspension Culture Grinding Buffer (1 liter)
0.3 M Mannitol ----------------------------------- 54.66 g
50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
3 mM EDTA ------------------------------------- 6 ml of 0.5 M EDTA
0.1% BSA --------------------------------------- 1 g
20 mM 2-mercaptoethanol ----------------------- 1.4 ml of 14.4
M 2-mercaptoethanol
Deionized H2O to make a final volume of -------- 1 liter
Gradient Buffer (1 liter)
0.3 M Sucrose ------------------------------------ 102.7 g
50 mM Tris-HCl (pH 8.0) ------------------------ 50 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 1 g
Deionized H2O to make a final volume of -------- 1 liter
1.6 M Sucrose Step Gradient Buffer (100 ml)
1.6 M Sucrose ------------------------------------ 54.8 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
1.2 M Sucrose Step Gradient Buffer (100 ml)
1.2 M Sucrose ------------------------------------ 41.1 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
0.6 M Sucrose Step Gradient Buffer (100 ml)
0.6 M Sucrose ------------------------------------ 20.5 g
50 mM Tris-HCl (pH 8.0) ------------------------ 5 ml of 1 M Tris-HCl
20 mM EDTA ------------------------------------ 40 ml of 0.5 M EDTA
0.1% BSA ---------------------------------------- 0.1 g
Deionized H2O to make a final volume of -------- 100 ml
ET buffer (100 ml)
20 mM EDTA ------------------------------------- 4 ml of 0.5 M EDTA
50 mM Tris-HCl (pH 8.0) ------------------------- 5 ml of 1 M Tris-HCl
Deionized H2O ------------------------------------ 91 ml
PROCEDURES
Isolation of Mitochondria
All steps should be performed in ice!
1. Grow suspension cultures in UMIA medium in dim light until
cultures appear white or light yellow and creamy. Such cultures
usually have lower amounts of phenolic compounds so that 2-mercaptoethanol
is sufficient and PVP can be omitted from the Suspension Culture
Grinding Buffer. If phenolics are present in damaging quantities,
1.0% PVP can be included during homogenization.
2. Break suspension cultures in a Bead-Beater with ice in outer jacket. Three times for 10 second-pulse at
high speed.
If using a French Press, break suspension cultures at 3,000 psi.
3. Collect 400-500g cells by centrifugation at 1,500g for 10 minutes at 4oC.
5. Pour the suspension into a Bead-Beater vessel containing one-half volume of 500-um glass beads.
6. Break suspension cultures in a Bead-Beater with ice in outer jacket. Three times for 10 second-pulse at
high speed.
7. Decant the disrupted cells from the glass beads into a Miracloth-lined
funnel.
8. Centrifuge the filtered preparation for 10 minutes at 1,500g to remove cell debris, plastids, and nuclei.
9. Transfer the supernatant to a new centrifuge tube. Centrifuge
at 15,000g for 15 minutes at 4oC.
10. Resuspend the brownish mitochondrial pellet in about 25 ml
of Suspension Culture Grinding Buffer.
11. Add 300 ul of 1 M MgCl2 and 250 ul of DNase I (10 mg/ml) to the crude mitochondrial suspension.
12. Incubate for 30 minutes on ice.
13. Add 1 ml 0.5 M EDTA to the incubation medium to stop the reaction.
14. Wash the mitochondria by diluting to about 400 ml with Gradient
Buffer and centrifuging at 15,000 g for 15 minutes.
15. Resuspend the pellet in 10 ml of Gradient Buffer.
16. Layer 3-4 ml of mitochondrial suspension per sucrose step
gradient consisting of 10 ml 1.6 M Sucrose Step Gradient Buffer,
10 ml 1.2 M Sucrose Step Gradient Buffer, and 10 ml 0.6 M Sucrose
Step Gradient Buffer.
17. Centrifuge for 1 hour at 25,000 rpm for purification from
contaminating subcellular structures and residual DNase.
18. A cream colored band will appear at both interfaces. Collect
only the lower band, which contains most of the mtDNA, with a syringe.
19. Dilute the mitochondrial fraction with 3 volumes of Gradient
Buffer slowly over a 15-minute period to minimize disruption by
osmotic shock.
20. Harvest the mitochondria by centrifugation at 15,000g for 10 minutes at 4oC.
21. Resuspend the mitochondrial pellet in 7 ml of ET buffer.
22. Add 350 ul of 10% Sarkosyl to lyse the mitochondria.
Optional; add 1 ul of Proteinase K (20 mg/ml) and incubate for 15 minutes on ice to reduce clumps
of debris.
CsCl Gradients for mtDNA Purification
23. To 7 ml of lysed mitochondria, add 8.05 g CsCl and 220 ul
of ethidium bromide (10 mg/ml).
24. Add ET buffer to make the total weight to 8.32 g. Dissolve
the CsCl.
25. Transfer the liquid to a Beckman Quick-Seal tube and heat-seal
the tube.
26. Centrifuge at 65,000 rpm for 8-10 hours at 20oC.
27. Collect DNA band with a syringe.
In some preparations, particularly with certain genotypes, a
lower band of supercoiled mtDNA is also present. Collect supercoiled
mtDNA, also.
28. Extract the ethidium bromide three times with isopropanol
equilibrated with CsCl-saturated TE buffer.
29. Dialyze mtDNA against 4 liters of STE buffer for 1-2 hour.
30. Add 1/10th volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of ethanol.
31. Precipitate DNA at -20oC overnight.
32. Collect DNA by centrifugation at 15,000 rpm at 4oC for 20 minutes.
33. Resuspend mtDNA in 100-500 ul TE and store at -20oC or 4oC.
NOTES
Deoxyribonuclease (DNase) is used to remove remaining DNA external to the mitochondria. The effectiveness of this DNase step requires
penetration of the DNase into nonintact contaminating plastids
and nuclear debris and sufficient mitochondrial integrity to prevent
the enzyme from entering the organelles. Integrity of mitochondria
is critical when utilizing a DNase step during purification. This
treatment should be performed early in the isolation procedure
while the highest proportion of mitochondria are intact.
In some mtDNA preparations, faint bands which comigrate with purified
plastid DNA restriction fragments can be seen on ethidium bromide-stained
gels of restriction enzyme-digested mtDNA preparations. Whether
these represent plastid DNA contamination can be checked by hybridizing
total plastid DNA (nick-translated) to a Southern blot.
If DNase treatment of sucrose gradient-purified fractions does
not give adequate purification from plastid DNA, dyes which enhance
separation of organelle DNAs can be incorporated into the CsCl
gradients. Plastid DNA could be quantitatively separated from
mtDNA on CsCl gradients containing diamidinophenylindol or bisbenzimide.
KIT INFORMATION
REFERENCES
Boeshore, ML, Lifshitz, I, Hanson, MR, Izhar, S (1983)
Mol. Gen. Genet. 190: 459.