PKbuffer.qmd

# Procedure to Make Proteinase K (PK) Buffer GE-002.00 {.unnumbered}
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.

**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.Use respirators if needed.\

**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:\\Users\ljuser\Desktop\MMTD Genetics 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]{.underline} \
*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]{.underline}\
*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]{.underline} \
*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.\