Standard Operating Procedure
Title:Transforming Competent Cells with Plasmid DNA
Department: Agronomy
Created by: Haiyan Huang
Laboratory: Crop Production & Physiology Lab Suite
Supervisor: Dr.Jianming Yu
Lab Supervisor: Whitney Bouma
Date approved:
Procedure Overview:
Transforming Competent Cells with Plasmid DNA
Equipment and reagents necessary:
Equipment: Water-bath; Incubator;
Reagents: Chemically Competent cells; SOC medium; Sodium chloride (NaCl); Tryptone; Yeast Extract;
Agar; Deoxyribonucleic acid (DNA); Ampicillin, sodium salt (Ampicillin); X-Gal - IPTG Ready
Solution.
Miscellaneous: Tips (10ul, 200ul, 1000ul); Tube (1.5ml tube); Culture plate; 0.22um Sterile Filters;
Syringe; Glass bead;
Procedure:
Solution preparation:
Ampicillin (100mg/ml)
10ml
Ampicillin
1g
Deionized water
add water to 10ml
Sterilize With a 0.22 um Sterile Filters
LB medium agar
Tryptone
Yeast extract
NaCl
Agarose
Deionized water
1000ml
10g
5g
10g
15g
add water to 1000ml
1. Transfer Chemically Competent cells from –70 oC refrigerator onto bench top to thaw on ice.
3. After mixing gently, aliquot 25ul of cells into chilled micro tube.
4. Add 1.25ul or less plasmid DNA (the volume of DNA solution should not exceed 5% of the volume of
the competent cells or no more than 50ng in a volume of 10ul or less).
5. Gently swirl tube(s) for a few seconds to mix by finger flicking tube.
6. Then incubate tube on ice for 30 minutes.
7. Heat shock tube(s) by placing in 42 oC water bath for ~45 seconds without shaking.
8. Replace tube(s) on ice for 2-5 minutes.
9. Add 900ul of SOC medium.
10. Gently shake and incubate tube(s) at ~200 rpm for 30-60 minutes at 37 oC.
11. Spread on LB agar plates containing appropriate antibiotic and X-gal Solution.
12. Place in the 37 oC incubator with the agar side up and the lid side down. Incubate overnight and
transfer to fridge (4 oC) the next day.
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
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Insulated gloves
Eye wash station
Safety shower
Respirator
Note: Open-toed and heeled shoes are NOT allowed.
Other Control Measures:
Handling & Storage Precautions:
Chemically Competent cells: Store at -80 oC in an appropriately labeled container.
DNA: Store at -20 oC.
SOC medium:
Handling: Handle in accordance with good industrial hygiene and safety practice.
Storage: Keep containers tightly closed in a dry, cool and well-ventilated place.
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
Tryptone:
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 breathe dust.
Storage: Keep container dry. Keep in a cool place. Ground all equipment containing material. Keep
container tightly closed. Keep in a cool, well-ventilated place. Combustible materials should be
stored away from extreme heat and away from strong oxidizing agents.
Yeast Extract:
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 breathe dust.
Storage: Keep container dry. Keep in a cool place. Ground all equipment containing material. Keep
container tightly closed. Keep in a cool, well-ventilated place. Combustible materials should be
stored away from extreme heat and away from strong oxidizing agents.
Agar:
Precautions: Use with adequate ventilation. Minimize dust generation and accumulation. Avoid contact
with skin and eyes. Keep container tightly closed. Avoid ingestion and inhalation. Wash
clothing before reuse.
Storage: Keep out of oxidizing materials. Store in a cool and dry, well-ventilated area away from
incompatible substances.
Ampicillin: Keep container tightly closed. Keep container in a cool, well-ventilated area.
X-Gal - IPTG Ready Solution:
Handing: Handle in accordance with good industrial hygiene and safety practice. Light Sensitive. Heat
Sensitive. Hygroscopic.
Storage: Keep containers tightly closed in a dry, cool and well-ventilated place. Protect from light. Keep
away from heat. Hygroscopic.
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.
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Spill/Release Containment and Clean Up/Decontamination Procedures:
SOC medium: Soak up with an inert absorbent material.
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.
Tryptone:
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.
Yeast Extract:
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.
Agar: Vacuum or sweep up material and place into a suitable disposal container. Clean up spill
immediately, observing precautions in the protective equipment section. Avoid generally
dusty conditions. Provide ventilation.
Ampicillin:
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.
X-Gal - IPTG Ready Solution: Dispose of material in accordance with all federal, state, and local
regulations.
Health & Safety Summary for Required Reagents:
NaCl: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of
inhalation.
Tryptone: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation (lung
irritant, lung sensitizer).
Yeast Extract: Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of
inhalation.
Agar: This is expected to be a low hazard for usual industrial handling. May cause eye, skin, and
respiratory tract irritation. Target Organs: None known
Ampicillin: Hazardous in case of ingestion, of inhalation. Slightly hazardous in case of eye contact
(irritant).
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X-Gal - IPTG Ready Solution: The product has to be labelled due to the calculation procedure of
international guidelines.Flammable.Irritating to eye, skin and respiratory system..
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Chemical name
NaCl
Tryptone
Yeast Extract
Agar
X-gal Solution
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Incompatibilities
1 0 0
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eye; skin
eye; skin
eye; skin
Spleen, Bone
marrow
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Strong acids.
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Ampicillin
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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:
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