Standard Operating Procedure
Title: DNA extraction from plants
Department: Agronomy
Created by: Haiyan Huang
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr.Jianming Yu
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
DNA extraction from plants
Equipment and reagents necessary:
Equipment: Centrifuge;
Freeze dryer;
Water-bath
Reagents: Sorbitol; Tris(hydroxymethyl)aminomethane (Tris base); Ethylenediaminetetraacetic Acid
Tetrasodium Salt (EDTA-Na2); Sodium chloride (NaCl); Cetyltrimethylammonium bromide
(CTAB); Liquid nitrogen; Chloroform/isoamyl (24:1); Isopropanol; Ethyl Alcohol (Ethanol);
Hydrochloric acid (HCl); Sodium Bisufite; Polyvinyl Pyrrolidone (PVP); RNase A (10mg/ml);
Sodium acetate (3M); Tris 1 Molar buffer
Miscellaneous: Tips (10ul, 200ul, 1000ul); Tube (2.0 ml); Weigh boats; Balance; Spatula
Procedure:
Buffer Preparation:
Extraction Buffer
500ml
0.35 M sorbitol
32g
0.1 M Tris base
6g
5mM EDTA-Na2
0.84g (or EDTA-Na4 1.0g)
deionized water
add water to 500ml
Adjust to pH 7.5 with high conc. HCl
Lysis Buffer
0.2M Tris-base
0.05M EDTA-Na2
2M NaCl
2% CTAB
1% PVP
deionized water
500ml
12.1g
8.4g (or EDTA-Na4 10g)
58.5g
10g
5g
add water to 500ml
Microprep Buffer (just before use)
20 ml Extraction Buffer
20 ml Lysis Buffer
0.2g Sodium bisulfite
1. Cut 0.2g tissue, add to 2.0 ml tube, freeze dry for 2 days, shake to power; or ground the tissue with
liquid nitrogen.
2. Add 650ul pre-heated (65°C) microprep buffer, gently mix, incubate at 65°C for 1 hr;
3. Add 650ul of chloroform/isoamyl (24:1), gently mix for 2 min by inverting the tube;
4. Centrifuge the tube at 10,000 rpm for 5 min;
5. Transfer aqueous phase (~600ul) into a new tube
6. Repeat 3, 4 and 5; add 400ul -20°C isopropanol, and mix well;
7. -20°C 20 min or overnight;
8. Spin at 10,000 rpm, 4 °C for 10 min;
9. Pour or pipette off the liquid, add 700ul cold 70% ethanol and mix;
10. Spin at 10,000 rpm, 4°C for 5 min;
11. Repeat 9 and 10;
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revised on 06-20-2013
12. Dry for 10-15 min upside down on a clean paper towel;
13. Re-suspend the DNA in 500ul H2O at room temperature or 4°C for 30 min;
14. Cool down to 37°C, add 2 ul RNase A, incubate at 37°C for 1 hr;
15. Add 500ul chloroform/isoamyl (24:1), gently mix by inversion, spin at 10,000 rpm for 10 min, transfer
the aqueous phase to a new tube;
16. Add 2 volumes of ice cold 100% ethanol combined with 1/10 volume sodium acetate (3M).
17. Store at -20°C for 30 min;
18. Spin at 10,000 rpm for 15 min;
19. Pour or pipette off the liquid, add 700ul cold 70% ethanol and mix well;
20. Spin at 10,000 rpm for 5 min;
21. Dry for 10-15 min upside down on a clean paper.
22. Re-suspend the DNA in 50ul H2O or Tris 1 Molar buffer at room temperature or 4°C for 30 min;
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.
Other Control Measures:
Handling & Storage Precautions:
Sorbitol:
Handling: Do not get water inside containers. Avoid contact with skin and eyes. Avoid ingestion and
inhalation. Use with adequate ventilation. Do not allow contact with water. Keep from contact
with moist air and steam.
Storage: Store in a tightly closed container. Store in a cool, dry, well-ventilated area away from
incompatible substances.Store protected from moisture.
Tris base:
Handling: Avoid contact with skin and eyes. Avoid formation of dust and aerosols.Provide appropriate
exhaust ventilation at places where dust is formed. Normal measures for preventive fire
protection.
Storage: Keep container tightly closed in a dry and well-ventilated place.
EDTA-Na2:
Precautions: Keep away from heat. Keep away from sources of ignition. Ground all equipment
containing material. Do not ingest. Do not breathe dust. If ingested, seek medical advice
immediately and show the container or the label. Keep away from incompatibles such as
oxidizing agents, metals.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.
NaCl:
Precautions: Keep locked up.. Do not ingest. Do not breathe dust. Avoid contact with eyes. Wear
suitable protective clothing. If ingested, seek medical advice immediately and show the
container or the label. Keep away from incompatibles such as oxidizing agents, acids.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area. Hygroscopic
CTAB:
Precautions: Keep away from heat. Keep away from sources of ignition. Ground all equipment
containing material. Do not ingest. Do not breathe dust. Wear suitable protective clothing.
In case of insufficient ventilation, wear suitable respiratory equipment. If ingested, seek
medical advice immediately and show the container or the label. Avoid contact with skin
and eyes. Keep away from incompatibles such as oxidizing agents.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.
Liquid nitrogen:
Handing: Never allow any unprotected part of the body to touch uninsulated pipes or vessels which
contain cryogenic fluids. The extremely cold metal will cause the flesh to stick
Storage: Store and use with adequate ventilation. Do not store in a confined space. Cryogenic
containers are equipped with pressure relief devices to control internal pressure. Under normal
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revised on 06-20-2013
conditions these containers will periodically vent product. Do not plug, remove, or tamper with
pressure relief device..
Chloroform/isoamyl (24:1): Store in a cool dry place, tightly closed, protected from light.
Isopropanol:
Precautions: Keep away from heat. Keep away from sources of ignition. Ground all equipment
containing material. Do not ingest. Do not breathe gas/fumes/ vapor/spray. Avoid contact
with eyes. Wear suitable protective clothing. In case of insufficient ventilation, wear suitable
respiratory equipment. If ingested, seek medical advice immediately and show the
container or the label. Keep away from incompatibles such as oxidizing agents, acids.
Storage: Store in a segregated and approved area. Keep container in a cool, well-ventilated area. Keep
container tightly closed and sealed until ready for use. Avoid all possible sources of ignition
(spark or flame).
Ethanol:
Precautions: Keep locked up. Keep away from heat. Keep away from sources of ignition. Ground all
equipment containing material. Do not ingest. Do not breathe gas/fumes/ vapor/spray.
Wear suitable protective clothing. In case of insufficient ventilation, wear suitable
respiratory equipment. If ingested, seek medical advice immediately and show the
container or the label. Avoid contact with skin and eyes. Keep away from incompatibles
such as oxidizing agents, acids, alkalis, moisture.
Storage: Store in a segregated and approved area. Keep container in a cool, well-ventilated area. Keep
container tightly closed and sealed until ready for use. Avoid all possible sources of ignition
(spark or flame). Do not store above 23°C (73.4°F).
RNase A:
Handling: Wash thoroughly after handling. Use with adequate ventilation. Minimize dust generation and
accumulation. Avoid contact with eyes, skin, and clothing. Keep container tightly closed. Avoid
ingestion and inhalation.
Storage: Store in a tightly closed container. Store in a cool, dry, well-ventilated area away from
incompatible substances. Store at -20°C.
HCl:
Precautions: Keep locked up.. Keep container dry. Do not ingest. Do not breathe gas/fumes/
vapor/spray. Never add water to this product. In case of insufficient ventilation, wear
suitable respiratory equipment. If ingested, seek medical advice immediately and
show the container or the label. Avoid contact with skin and eyes. Keep away from
incompatibles such as oxidizing agents, organic materials, metals, alkalis, moisture.
May corrode metallic surfaces. Store in a metallic or coated fiberboard drum using a
strong polyethylene inner package.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.
Sodium Bisufite:
Precautions: Keep locked up.. Keep container dry. Do not ingest. Do not breathe dust. Never add water
to this product. In case of insufficient ventilation, wear suitable respiratory equipment. If
ingested, seek medical advice immediately and show the container or the label. Avoid
contact with skin and eyes. Keep away from incompatibles such as oxidizing agents,
alkalis.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.
Polyvinyl Pyrrolidone (PVP)
Handling:Wash thoroughly after handling. Remove contaminated clothing and wash before reuse. Use
with adequate ventilation. Minimize dust generation and accumulation. Avoid contact with
eyes, skin, and clothing. Keep container tightly closed. Avoid ingestion and inhalation.
Storage: Store in a tightly closed container. Store in a cool, dry, well-ventilated area away from
incompatible substances.
Sodium acetate (3M):
Handling: Precautions. Keep away from heat. Keep away from sources of ignition. Empty containers
pose a fire risk, evaporate the residue under a fume hood. Ground all equipment
containing material. Do not ingest. Do not breathe dust. Wear suitable protective clothing.
In case of insufficient ventilation, wear suitable respiratory equipment. If ingested, seek
medical advice immediately and show the container or the label. Keep away from
incompatibles such as oxidizing agents, acids.
Storage: Keep container tightly closed. Keep container in a cool, well-ventilated area.
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revised on 06-20-2013
Tris 1 Molar Buffer:
Handling: Wash thoroughly after handling. Remove contaminated clothing and wash before reuse. Use
with adequate ventilation. Minimize dust generation and accumulation. Avoid contact with
skin and eyes. Keep container tightly closed. Avoid ingestion and inhalation. Discard
contaminated shoes.
Storage: Store in a tightly closed container. Store in a cool, dry, well-ventilated area away from
incompatible substances.
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:
Sorbitol: Vacuum or sweep up material and place into a suitable disposal container. Clean up spills
immediately, observing precautions in the Protective Equipment section. Avoid generating
dusty conditions. Do not get water inside containers.
Tris base: Vacuum or sweep up material and place into a suitable disposal container. Avoid
generating dusty conditions. Provide ventilation. Do not let this chemical enter the
environment.
EDTA-Na2:
Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container.
If necessary: Neutralize the residue with a dilute solution of acetic acid. Finish cleaning
by spreading water on the contaminated surface and dispose of according to local and
regional authority requirements.
Large Spill: Use a shovel to put the material into a convenient waste disposal container. Neutralize
the residue with a dilute solution of acetic acid. Finish cleaning by spreading water on
the contaminated surface and allow to evacuate through these anitary system.
NaCl:
Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container.
Finish cleaning by spreading water on the contaminated surface and dispose of
according to local and regional authority requirements.
Large Spill: Use a shovel to put the material into a convenient waste disposal container. Finish
cleaning by spreading water on the contaminated surface and allow to evacuate
through the sanitary system.
CTAB:
Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container.
Finish cleaning by spreading water on the contaminated surface and dispose of
according to local and regional authority requirements.
Large Spill:Use a shovel to put the material into a convenient waste disposal container. Finish
cleaning by spreading water on the contaminated surface and allow to evacuate
through the sanitary system.
Chloroform/isoamyl (24:1): Ventilate area and wash spill site after material pickup is complete.
Isopropanol:
Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an
appropriate waste disposal container.
Large Spill: Flammable liquid. Keep away from heat. Keep away from sources of ignition. Stop leak if
without risk. Absorb with DRY earth, sand or other non-combustible material. Do not touch
spilled material. Prevent entry into sewers, basements or confined areas; dike if needed. Be
careful that the product is not present at a concentration level above TLV. Check TLV on the
MSDS and with local authorities.
Ethanol:
Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an
appropriate waste disposal container.
Large Spill: Flammable liquid. Keep away from heat. Keep away from sources of ignition. Stop leak if
without risk. Absorb with DRY earth, sand or other non-combustible material. Do not touch
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revised on 06-20-2013
spilled material. Prevent entry into sewers, basements or confined areas; dike if needed. Be
careful that the product is not present at a concentration level above TLV.
RNase A: observing precautions in the Protective Equipment section. Avoid generating dusty conditions.
Provide ventilation.
HCl:
Small Spill: Dilute with water and mop up, or absorb with an inert dry material and place in an
appropriate waste disposal container. If necessary: Neutralize the residue with a dilute
solution of sodium carbonate.
Large Spill: Corrosive liquid. Poisonous liquid. Stop leak if without risk. Absorb with DRY earth, sand
or other non-combustible material. Do not get water inside container. Do not touch spilled
material. Use water spray curtain to divert vapor drift. Use water spray to reduce vapors.
Prevent entry into sewers, basements or confined areas; dike if needed. Call for
assistance on disposal. Neutralize the residue with a dilute solution of sodium carbonate.
Be careful that the product is not present at a concentration level above TLV. Check TLV
on the MSDS and with local authorities.
Sodium Bisufite:
Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container.
Large Spill: Corrosive solid. Stop leak if without risk. Do not get water inside container. Do not touch
spilled material. Use water spray to reduce vapors. Prevent entry into sewers, basements
or confined areas; dike if needed. Call for assistance on disposal.
Polyvinyl Pyrrolidone (PVP): Vacuum or sweep up material and place into a suitable disposal container.
Clean up spills immediately, observing precautions in the Protective Equipment section.
Avoid generating dusty conditions. Provide ventilation.
Sodium Acetate (3M)
Small Spill: Use appropriate tools to put the spilled solid in a convenient waste disposal container. If
necessary: Neutralize the residue with a dilute solution of acetic acid. Finish cleaning by
spreading water on the contaminated surface and dispose of according to local and regional
authority requirements.
Large Spill: Use a shovel to put the material into a convenient waste disposal container. Neutralize the
residue with a dilute solution of acetic acid.
Tris 1 Molar Buffer: Absorb spill with inert material (e.g. vermiculite, sand or earth), then place in suitable
container. Avoid runoff into storm sewers and ditches which lead to waterways. Clean up
spills immediately, observing precautions in the Protective Equipment section. Provide
ventilation.
Health & Safety Summary for Required Reagents:
Sorbitol: Hazardous in case of eye contact (irritant), of ingestion. Slightly hazardous in case of skin
contact (irritant), of inhalation.
Tris base: For Inhalation may be harmful if inhaled. Causes respiratory tract irritation. For Skin may be
harmful if absorbed through skin. Causes skin irritation and eye irritation.
EDTA-Na2: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion,
ofinhalation.
NaCl: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of
inhalation.
CTAB: Hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation.
Liquid nitrogen: Exposure to an oxygen deficient atmosphere (<19.5%) may cause dizziness, drowsiness
nausea,vomiting, excess salivation, diminished mental alertness, loss of consciousness and
death. Exposure to atmospheres containing 8-10% or less oxygen will quickly bring about
unconsciousness without warning, leaving individuals unable to help or protect themselves. Lack
of sufficient oxygen can cause serious injury or death.Skin contact with liquid nitrogen can cause
tissue freezing, resulting in severe burns. The burns are caused by the extremely low
temperature of the cryogenic liquid and not the result of chemical action. Skin may appear red
with the formation of blisters. In cases that involve prolonged or severe exposure, tissue may
freeze and have a waxy or yellow appearance.
Chloroform/isoamyl (24:1): Harmful if swallowed. Irritating to eyes and skin. Limited evidence of a
carcinogenic effect. Harmful: danger of serious damage to health by prolonged exposure
through inhalation and if swallowed.
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revised on 06-20-2013
Isopropanol: Hazardous in case of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in
case of skin contact (irritant,sensitizer, permeator).
Ethanol: Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Slightly
hazardous in case of skin contact (permeator), of ingestion.
HCl: Very hazardous in case of skin contact (corrosive, irritant, permeator), of eye contact (irritant,
corrosive), of ingestion, . Slightly hazardous in case of inhalation (lung sensitizer). Non-corrosive
for lungs. Liquid or spray mist may produce tissue damage particularly on mucous membranes of
eyes, mouth and respiratory tract. Skin contact may produce burns. Inhalation of the spray mist
may produce severe irritation of respiratory tract, characterized by coughing, choking, or
shortness of breath. Severe over-exposure can result in death. Inflammation of the eye is
characterized by redness, watering, and itching. Skin inflammation is characterized by itching,
scaling, reddening, or, occasionally, blistering.
Sodium Bisufite: Very hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of
inhalation. Hazardous in case of skin contact (corrosive, permeator). The amount of tissue
damage depends on length of contact. Eye contact can result in corneal damage or blindness.
Skin contact can produce inflammation and blistering. Inhalation of dust will produce irritation to
gastrointestinal or respiratory tract, characterized by burning, sneezing and coughing. Severe
over-exposure can produce lung damage, choking, unconsciousness or death. Inflammation of
the eye is characterized by redness, watering, and itching. Skin inflammation is characterized by
itching, scaling, reddening, or, occasionally, blistering.
Polyvinyl Pyrrolidone (PVP): The toxicological properties of this material have not been fully investigated.
May cause eye and skin irritation. May cause respiratory and digestive tract irritation. Target
Organs: No data found.
Sodium Acetate (3M): Hazardous in case of ingestion, of inhalation. Slightly hazardous in case of skin
contact (irritant), of eye contact (irritant).
Tris 1 Molar Buffer: May cause eye and skin irritation. May cause respiratory and digestive tract irritation.
The toxicological properties of this material have not been fully investigated. Target Organs: None
known.
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Chemical name
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Organ(s)
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×
EDTA-Na2
×
NaCl
×
CTAB
Liquid nitrogen
×
×
×
Isopropanol
× × × × ×
RNase A
HCl
×
Sodium Bisufite
PVP
Sodium Acetate
Tris 1 Molar
Buffer
Blood. Central
nervous system.
Cardiovascular
system. Liver.
Kidneys
Gastrointestinal
tract, Liver,
Cardiovascular
system., Kidney,
Nerves
Kidneys, heart,
central nervous
system, liver
kidneys
×
×
×
×
x
Strong oxidizing agents,
bases, aluminum, brass,
copper
Reactive with oxidizing
agents, metals
Reactive with oxidizing
agents, metals, acids
Reactive with oxidizing
agents
Strong bases, strong
oxidizing agentes,
Aluminum, Magnesium,
Sodium, Lithium
Sensitive to light
oxidizing agents, acids,
alkalis.
oxidizing agents, acids,
alkalis.
× metals. Reactive with
×
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Incompatibilities
Respiratory
system, eyes, skin
Tris base:
Ethanol
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Sorbitol:
Chloroform/isoam
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oxidizing agents, organic
materials, alkalis, water
oxidizing agents, alkalis
x
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 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:
7
revised on 06-20-2013
2 1 0
2 0 0
1 1 0
1 0 0
2 1 0
3 0 0
2 0 1
2 3 1
2 3 0
3 0 1
3
1
2
1
0
0
1
0
0
0
0
0