Standard Operating Procedure
Title: Mitochondrial Isolation from Etiolated Corn Shoots
Department: Agronomy
Created by: Chris Hejlik
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr. Allen Knapp
Lab Supervisor: Whitney Bouma
Date Approved:__________________
Procedure Overview:
The procedure provided is for the mitochondrial isolation of etiolated corn shoots after
approximately seven days. Isolated mitochondria are to be used for gradient purification,
integrity checks by ADP/O ratio measurements, as well as respiration measurements.
MSDS Information:
MSDS Sheets:
Available for reference in laboratory 1522
Chemicals:
Mannitol
MOPS
EGTA
BSA
Cysteine
KOH
Equipment and reagents necessary:
Plant Material(s)
1) Etiolated Seedlings grown from the following lots
a. M808 E-10 VT3 RRs seed lots, 108 days maturity
i. Seed lot treated with active ingredient and fillers
ii. Seed lot treated with fillers
iii. Seed lot with no treatment
b. M314 A-10 VT3 RR2 seed lots, 114 days maturity
i. Seed lot treated with active ingredient and fillers
ii. Seed lot treated with fillers
iii. Seed lot with no treatment
Equipment
1) Waring Blender
2) Multiple layers of cheesecloth Centrifuge, CP & P Lab
a. Centrifuge tubes
i. 503) Growth Chamber
4) Scissors or razor blade
5) Ruler
6) Various beakers
a. 50b. 100c. 250-ml
Reagents
1) Grinding Medium
a. 350 mM Mannitol
b. 30 mM MOPS
c. 1 mM EGTA
2) 8 M KOH
3) Wash Medium
a. 350 mM Mannitol
b. 20 mM MOPS
c. 1 mM EGTA
4) 0.84 g PVPP per trial
5) .1634 g cysteine per trial
Procedure
1. Cut etiolated corn shoots into segments 0.5 - 1.0 cm long using a razor blade; material
should include mesocotyls only
2. Grinding medium, cysteine, and PVPP added to Waring Blender.
a. Grinding medium added in a 3.33:1 ratio to starting fresh weight
3. Lid of Waring Blender added, and homogenization started
a. Homogenization should occur in short bursts to avoid breaking of mitochondria
cells
b. Short bursts include 3 reps x 5 seconds
4. Other options for homogenization, based on availability
a. Homogenized in square-form beaker with polytron at 50% full speed for 2 x 10
seconds
b. Pre-cooled mortar and pestle
5. Homogenate is filtered through two layers of cheesecloth into beakers cooled in ice.
Cheesecloth is squeezed to ensure removal of most of the fluid
6. pH of filtrate is adjusted to 7.2 - 7.5 by drop-wise addition of 8 M KOH using one of the
following
a. narrow range pH paper
b. pH meter
7. Filtrate divided equally into 50 ml conical bottom centrifuge tubes
a. Amount of tubes dependent on starting tissue and grinding medium
8. Centrifuge bottles are balanced and centrifuged for 2 minutes at 5200 g in a centrifuge
(CP&P Lab) at 2°C
9. Supernatant fraction decanted into clean, cold centrifuge bottles via pouring; do not
transfer any of the pellet
a. Pellet discarded in accordance with EH&S policies outlined in Waste Disposal
section
10. Supernatant in clean tubes spun at 19,250 g for 5 minutes
a. Resulting supernatant discarded via pouring into waste container
i. Supernatant discarded in accordance with EH&S policies outlined in
Waste Disposal Section
11. Crude mitochondria pellets washed with 5 ml of wash medium
a. Eppendorf micropipette, 500-5000µl size, is used
b. Pellet re-suspended via glass stirring rod
i. Leave behind small white mass (this is starch)
c. Pool the resulting crude mitochondrial solution into one tube per trial
i. Resulting small white starch discarded in accordance with EH&S policies
outlined in Waste Disposal section
12. Centrifuge at 5200 g for 2 minutes.
a. Pipette supernatant into clean 50 ml, conical bottom centrifuge tubes while
leaving behind slimy film
i. Eppendorf micropipette used
ii. Slimy film discarded in accordance with EH&S policies outlined in Waste
Disposal section
13. Underlay each centrifuge tube with 8 ml of sucrose media
a. Using an Eppendorf 5000 µl micropipette, draw 5 ml of sucrose solution
b. Insert pipette into centrifuge tube, all the way to the bottom
c. Dispense sucrose solution in an even motion, being careful to not press any air
bubbles into the liquid. Once all sucrose is dispensed, keep button depressed (to
not draw any liquid back up) while taking the pipette out of solution
d. Change the setting on the pipette to 3 ml, and repeat a - c
14. Centrifuge resulting sucrose cushion and wash medium at 10,400 g for 20 minutes
15. Aspirate supernatant, leaving behind mitochondrial pellet
16. Re-suspend mitochondrial pellet with suspension medium and place on ice for future use
Personal Protective Equipment / Engineering Controls:
Nitrile gloves
Safety glasses
Face shield
Dust mask
Latex gloves
Splash goggles
Lab coat
Fume hood
Neoprene gloves
Vented goggles
Apron
Biosafety cabinet
Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Skin - Wear protective, nitrile gloves and a laboratory coat. Avoid contact with skin and clothing. If
exposure to skin occurs, wash immediately with non-abrasive soap and water.
Eyes - Wear safety (or splash when using KOH) goggles to avoid exposure to eyes. If exposure to
eyes occurs, remove any contact lenses and immediately flush with water for 15 minutes.
If swallowed - do not induce vomiting. Give large quantities of water to dilute any/all chemicals.
Seek medical attention.
Handling & Storage Precautions:
Keep all containers tightly closed and in a dry, cool, well-ventilated area. Avoid sources of
ignition and flammable products. In addition, refer to MSDS(s) for each chemical’s
specific conditions.
Waste Disposal Procedures:
Unless EH&S specifically instructs otherwise, all chemical/reagent waste (including excess
solutions) must be placed in an appropriately labeled hazardous waste container for EH&S
disposal. Compatible substances may be combined into one waste container.
Spill/Release Containment and Clean Up/Decontamination Procedures:
Accidental Release - KOH: Absorb neutralized caustic residue on clay, vermiculite or other inert
substance and package in a suitable container for disposal. Try to avoid creating dust.
Accidental Release - ALL: Ventilate area of leak/spill, avoid ignition, clean and expose in
accordance with EH&S, and wash contaminated area with water.
Health & Safety Summary for Required Reagents:
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Chemical name
Mannitol
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EGTA
X X
MOPS
X X
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Target Organ(s)
May cause
irritation to eyes
and skin
Toxic in large
amounts by
ingestion
Very hazardous
in case of eye
contact; toxic to
lungs
BSA
Cysteine
X X
KOH
X X
2-mercapthoethanol
X X
Hazardous in
the case of
skin/eye
contact,
ingestion and
inhalation
Severe irritant of
eyes and skin.
Toxic
Toxic; harmful if
swallowed.
X
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Incompatibilities
Strong Oxidizers; avoid
heat, flames, and ignition
sources
1 1 0
Keep away from heat and
sources of ignition
2 1 0
Strong oxidizers and
bases; Keep away from
heat and sources of
ignition
2 1 0
Strong acids and
oxidizing agents
1 1 0
May be combustible at
high temps. Avoid
sources of ignition;
evaporate residue under
fume hood; avoid strong
oxidizers
1 1 0
Corrosive; avoid contact
with water, acids,
flammable liquids. Avoid
heat and moisture.
Oxidizing agents and
moisture. Avoid contact
with metals, heat, and
ignition sources.
3 0 2
 The above summary consists of guidelines for proper handling & disposal of chemicals
used in this procedure. You must read and understand the contents of the entire
MSDS(s) before starting this procedure.
References:
Influence of Trinexapac-Ethyl on Respiration of Isolated Wheat Mitochondria. Heckman,
Elthon, Horst and Gaussoin. Crop Science (2002) 42:2: 423-427
Acclimation, Hydrogen Peroxide, and Abscisic Acid Protect Mitochondria against
Irreversible Chilling Injury in Maize Seedlings. Prasad TK, Anderson MD, Stewart CR.
Plant Physiology (1994) 105: 619 - 627
MSDS sheets (on file)
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