General Lab (SOP’s)

A comprehensive list of Standard Operating Procedures (i.e. protocols) for use in the general lab can be found below.

GE-035.00

Procedure to Make 1x Low Tris-EDTA (TE) Buffer pH 7.5 (from solution) GE-035.00

SOP IDENTIFIER: GE-035.00
ORIGINAL SOP DATE: 7/20/2023
NUMBER OF PAGES IN SOP: 3
REVISION NUMBER: 00
REVISION DATE: N/A
WRITTEN BY: Erin LaCasella 7/20/2023
REVIEWED BY: Amy Frey
APPROVED BY: Victoria Pease
EFFECTIVE DATE:

Scope and Purpose:
To make 1X Low TE buffer for DNA storage. TE buffer solubilizes DNA or RNA for storage and protects from degradation and has a lower salt content. Ethylenediaminetetraacetic Acid (EDTA) chelates metal ions (e.g. Mg 2+) which normally cause degradation.

Summary of Method:
Buffer is made by combining pre-made solutions of: 1M Tris-HCl (pH 7.5) and 0.5 M Ethylenediaminetetraacetic Acid (EDTA, pH 8.0).

Health and Safety Warnings:
• This procedure uses chemicals that may be harmful. Please read Appendix A before performing this protocol.
• Wear gloves, lab coat, closed-toe shoes and safety glasses.

Quality Control:
• Store in the refrigerator in a tightly closed container
• Perform procedure in general lab

Equipment and Supplies:
1. 250 mL (or 500 mL) Graduated cylinder
2. 250 mL (or 500 mL) orange screw cap bottle
3. Tris-HCl (Invitrogen Ultrapure 1M Tris-HCl pH 7.5)
4. EDTA (Invitrogen Ultrapure 0.5M EDTA, pH8.0)
5. MilliQ water (or Invitrogen Ultrapure water)

Procedures: To make 250 mL, combine reagents in a graduated cylinder:
1. Add 2.5 mL of 1M Tris-HCl pH 7.5 (pre-made solution)
2. Add 50 uL of 0.5M EDTA pH 8.0 (pre-made solution)
3. Add MilliQ water to total 250 mL
4. Pour into the orange screw cap bottle
5. Mix the solution by carefully inverting the bottle a few times
6. Label the bottle and store in refrigerator

Waste Management: If there are questions on handling chemicals contact the Safety Officer or Lab Manager. Spills, cuts or other accidents should be reported to the Safety Officer and Lab Manager.

References: For SDS information, go to C:\UsersGenetics SDS (see Lab Manager for designated computers).

Hazardous substance information:
https://epa.gov/ceppo/pubs/title3.pdf

Appendix A: Chemical Health and Safety Warnings

TE Buffer (Tris and EDTA)
Can cause damage to the kidneys, central nervous system and reproductive systems. It can cause skin, eye, digestive tract and respiratory irritation as well.
First Aid
Eye contact: flush eyes with water for at least 15 minutes while occasionally opening and closing eyelids. Seek medical attention immediately.
Skin: wash skin with lots of water and soap. Wash clothes before wearing them again and see a doctor if further skin irritation occurs.
Ingestion: drink 2-4 cups of milk if conscious and then seek medical aid.
Inhalation: seek fresh air immediately. If user has trouble breathing, give him/her oxygen and seek medical attention.
Tris
Irritating to the eyes, skin, mucous membrane and the upper respiratory tract. Signs and symptoms of exposure include watery eyes and rough, red skin. When inhaled, it can cause irritation, cough and shortness of breath. Exposing the substance into excessive heat may cause thermal decomposition and produce toxic fumes like carbon monoxide. It is incompatible with oxidizing materials.
First Aid
Eyes: flush with water for 15 minutes.
Skin: flush with water and wash thoroughly with soap and water. Remove contaminated clothing. If irritation persists, seek medical advice.
Inhalation: move victim to fresh air.
Ingestion: drink water and seek immediate medical attention.
EDTA
Irritating to the eyes, skin and the mucous membrane of the upper respiratory tract. It is flammable –do not expose to heat or flame. Flame caused by EDTA can be extinguished by carbon dioxide, foam, water or dry chemicals. EDTA has emerged as a persistent organic pollutant.
First Aid
Skin: rinse with water immediately
Eye contact: wash with water immediately and contact a physician right away.

GE-001.00

Procedure to Make 25X TBE Buffer GE-001.00

SOP IDENTIFIER: GE-001.00
ORIGINAL SOP DATE: 9/01/2008
NUMBER OF PAGES IN SOP: 5
REVISION NUMBER: 00
REVISION DATE: N/A
WRITTEN BY: Louella Dolar 9/01/2008
REVIEWED BY: Amy Frey 10/01/2008
APPROVED BY: Victoria Pease 4/27/2023
EFFECTIVE DATE: 09/25/2018

Scope and Purpose:
This SOP describes the process to make 25X TBE buffer to be used for electrophoresis.

Summary of Method:
Buffer is made by combining Tris base, Boric Acid and Ethylenediaminetetraacetic Acid (EDTA); then dissolving them in MilliQ water.

Quality Control:
• Store in the refrigerator in a tightly closed container
• Perform procedure in general lab

Equipment and Supplies:
1. Funnel
2. Reagent scale
3. Graduated cylinder
4. 0.47um HA Millipore filter paper
5. Filter/stopper for flask
6. Side arm filter flask
7. 2-1L orange screw cap bottle
8. Vacuum pump
9. Stir plate
10. Stir bar
11. Tris Base (non HCL, biological/molecular grade)
12. Boric Acid (biological/molecular grade)
13. EDTA (Ethylenediaminetetraacetic Acid, biological/molecular grade)

Procedures:
To make 1L:
1. Combine:
• 270.0g Tris Base (non HCl)
• 137.5g Boric Acid
• 20.75g EDTA
2. Bring to 950ml with MilliQ water.
3. Place stir bar in bottle and place cap on loosely. Place the bottle on the stir plate and set the stir plate to low heat and medium to high speed.
4. Stir until fully dissolved (approximately 2 hours with heat). 5. Bring to 1000ml with MilliQ water.
6. Attach the vacuum pump to the side arm filter flask.
7. Put filter paper on the filter (between the two pieces) and place the filter/stopper in the flask, creating a seal for the vacuum.
8. Filter small portions of solution at a time, changing the filter every other time.
9. Transfer to a 1L orange screw cap bottle.
10. Refer to SOP GE-023.00 for pH protocol. pH should be between 8.0-8.4 at room temperature.
11. Autoclave for 20 minutes to keep TBE in solution.
12. Store at room temperature.
13. Discard when precipitate appears on bottom of bottle.
Waste Management: If there are questions on handling chemicals contact the Safety Officer or Lab Manager. Spills, cuts or other accidents should be reported to the Safety Officer and Lab Manager.

References: For SDS information, go to C:\UsersGenetics SDS (see Lab Manager for designated computers).

Hazardous substance information:
https://epa.gov/ceppo/pubs/title3.pdf

Appendix A: Chemical Health and Safety Warnings

TBE Buffer (Tris, Boric Acid, and EDTA)
Can cause damage to the kidneys, central nervous system and reproductive systems. It can cause skin, eye, digestive tract and respiratory irritation as well.
First Aid
Eye contact: flush eyes with water for at least 15 minutes while occasionally opening and closing eyelids. Seek medical attention immediately.
Skin: wash skin with lots of water and soap. Wash clothes before wearing them again and see a doctor if further skin irritation occurs.
Ingestion: drink 2-4 cups of milk if conscious and then seek medical aid.
Inhalation: seek fresh air immediately. If user has trouble breathing, give him/her oxygen and seek medical attention.
Tris
Irritating to the eyes, skin, mucous membrane and the upper respiratory tract. Signs and symptoms of exposure include watery eyes and rough, red skin. When inhaled, it can cause irritation, cough and shortness of breath. Exposing the substance into excessive heat may cause thermal decomposition and produce toxic fumes like carbon monoxide. It is incompatible with oxidizing materials.
First Aid
Eyes: flush with water for 15 minutes.
Skin: flush with water and wash thoroughly with soap and water. Remove contaminated clothing. If irritation persists, seek medical advice.
Inhalation: move victim to fresh air.
Ingestion: drink water and seek immediate medical attention.
Boric Acid
Hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Slightly hazardous in case of skin contact (permeator). Boric Acid may be harmful to reproductive systems. The substance can be toxic to kidneys, cardiovascular systems and the central nervous system.
First Aid
Eye contact: In case of contact, immediately flush eyes with plenty of water for at least 15 minutes. Cold water may be used. Get medical attention. Skin: In case of contact, immediately flush skin with plenty of water. Cover the irritated skin with an emollient. Remove contaminated clothing and shoes. Cold water may be used. Wash clothing before reuse. Thoroughly clean shoes before reuse. Get medical attention. Wash with a disinfectant soap and cover the contaminated skin with an anti-bacterial cream. Seek medical attention. Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is difficult, give oxygen. Seek medical attention if serious inhalation.
EDTA
Irritating to the eyes, skin and the mucous membrane of the upper respiratory tract. It is flammable –do not expose to heat or flame. Flame caused by EDTA can be extinguished by carbon dioxide, foam, water or dry chemicals. EDTA has emerged as a persistent organic pollutant.
First Aid
Skin: rinse with water immediately
Eye contact: wash with water immediately and contact a physician right away.

GE-002.00

Procedure to Make Proteinase K (PK) Buffer GE-002.00

SOP IDENTIFIER: GE-002.00
ORIGINAL SOP DATE: 6/27/2011
NUMBER OF PAGES IN SOP: 4
REVISION NUMBER: 00
REVISION DATE: N/A
WRITTEN BY: Gabriela Serra-Valente
REVIEWED BY: Amy Jue
APPROVED BY: Victoria Pease 3/6/2019
EFFECTIVE DATE:

Scope and Purpose:
To make Proteinase K (PK) buffer to be used for making PK solution (see SOP PR-014.00).

Summary of Method:
Buffer is made by combining Tris-HCL, Glycerol and CaCl and dissolving them in MilliQ water.

Quality Control:
• Wear latex or nitrile gloves, closed-toe shoes and a lab coat.
• Wipe down surface area and pipettes with bleach/EtOH mixture to sterilize surfaces before and after use.
• Make sure all equipment has been properly cleaned and sterilized.
• Store at room temperature in tightly closed containers.
• Perform procedure in general lab.

Equipment and Supplies:
• Bench Paper
• Gloves
• One 250ml orange capped bottle, sterile
• Reagent scale
• One 100ml graduated cylinder
• Plastic weighing dish
• Scoopula/spatula
• Pipette (1000ul)
• Pipette tips (1000ul)
• Stir plate
• Stir bar
• Stir spoon
• 1M Tris-HCL, pH8.0 (see SOP GE-020.00)
• Glycerol
• Calcium Chloride (solid CaCl2)
• MilliQ water

Procedures:
To make 100ml:
1. Measure and add to the bottle:
• 50ml of Glycerol
• 1ml of 1M Tris-HCL, pH8.0
• 0.29g of CaCl2
2. Bring solution in the bottle to 100ml with MilliQ water.
3. Place stir bar in bottle, and place bottle on a stir plate; set the stir plate on low speed, no heat.
4. Stir until solution is thoroughly mixed and CaCl2 is fully dissolved.
5. Remove bottle from stir plate and retrieve stir bar with stir spoon.
6. Store at room temperature.
7. Label bottle with date and initials.
8. Clean all instruments used.

Waste Management: If there are questions on handling chemicals contact the Safety Officer or Lab Manager. Spills, cuts or other accidents should be reported to the Safety Officer and Lab Manager.

References: For SDS information, go to C:\UsersGenetics SDS (see Lab Manager for designated computers).

Hazardous substance information:
https://epa.gov/ceppo/pubs/title3.pdf

Appendix A: Chemical Health and Safety Warnings

Calcium Chloride (CaCl2)
Irritating to eyes, skin and respiratory tract. Dust-like, avoid breathing in.
First Aid
Eye contact: Flush immediately with water.
Skin: Flush immediately with water.
Inhalation: seek fresh air immediately.

Glycerol
Irritating to eyes, skin and respiratory tract. Wear appropriate personal protection equipment when handling this compound.
First Aid
Eyes: flush immediately with water.
Skin: flush immediately with water.
Inhalation: seek fresh air immediately.

Tris HCL
Irritating to the eyes, skin, mucous membrane and the upper respiratory tract. When inhaled, it can cause irritation, cough and shortness of breath. Chronic ingestion can cause nausea, vomiting and diarrhea; large amounts can cause weakness, collapse and coma. Exposing the substance to excessive heat may cause thermal decomposition and produce toxic fumes like carbon monoxide.
First Aid
Eye contact: flush with water for 15 minutes
Skin: flush with water and wash thoroughly with soap and water. Remove contaminated clothing. Get medical attention if irritation persists. Inhalation: remove victim from area of exposure and move him/her to fresh air.
Ingestion:drink water and seek immediate medical attention.