Sample Biological Material Standard Operating Procedure Template

SOP Polymerase Chain Reaction (PCR) Starr Lab

Title or Type of Procedure: Polymerase Chain Reaction (PCR)

P. I. Timothy K. Starr Lab Location: 12-175 MoosT

Original Issue Date: 5/21/11 Revision Dates: 3/4/14

Prepared By: Timothy K. Starr Approval Signature: (if required by lab supervisor)

Procedural Methods and Materials: General safety precautions Wear Personal Protective Equipment (PPE): gloves and lab coat. Handle ethidium bromide carefully, dispose as hazardous waste

Procedural Methods and Materials: Note: Keep all ingredients and sample tubes on ice as much as possible. Carry ice bucket to PCR machines. 1) Fill out the PCR excel spreadsheet and printout the sheet. 2) Sign-up for a PCR machine and make sure the correct PCR program exists on the machine. 3) Thaw DNA samples and controls and place on ice 4) Gather PCR cocktail ingredients (except for the taq) and PCR primers and put on ice. Make sure you have all the ingredients you need including sufficient amounts of primers. 5) Label PCR tubes or plates and place on ice in a 96 tube rack. 6) Aliquot DNA samples and controls (usually 1 or 2 µl containing between 100 to 500 ng DNA) into PCR tubes or plates. Touch pipette tip to bottom of tube when aliquoting, so DNA is at the bottom of the tube. 7) Mix the PCR cocktail using the amounts listed on the PCR excel spreadsheet in a 1.5 ml microcentrifuge tube. Always add the Taq polymerase last. Mix the complete cocktail by pipetting up and down. The Taq polymerase is normally in a glycerol solution, so it needs to be mixed well. 8) Aliquot the PCR cocktail into the PCR tubes or plates. If pipette tip touches a tube, throw it away and use a clean one. 9) Put caps on the PCR tubes or plates and seal completely. 10) Spin PCR tubes/plates. 11) Place PCR tubes/plates in PCR machine and start the PCR program. 12) Store PCR tubes/plates at 4ºC until they are analyzed on an agarose gel. Agarose Gel Analysis of PCR product 1) Prepare gel tray by either taping ends or putting rubber-edged acrylic end pieces on tray. Place a sufficient number of well combs in tray to handle all of your samples. Make sure well combs are spaced sufficiently to allow separation of DNA bands. 2) Fill Erlenmeyer flask with TAE buffer. Amount of TAE buffer will depend on the size of the gel tray. Use the following table for the amount of TAE buffer to use for the GeneMate gel trays: Gel Tray TAE (ml) Small 80 Medium 160 Large 250 X-Large 500 3) Add Agarose (Low EEO grade) to Erlenmeyer flask. Amount of Agarose will depend on the size of the DNA fragments that you are analyzing. Smaller DNA fragments require a higher percentage of agarose in the gel. Use the following table to determine what percentage of agarose to use: DNA size (bp) Agarose % (w/v) >1,000 0.6 (can go as low as 0.3 if DNA is >5,000 bp) 200 - 1,000 1.0 50 - 200 2.0 (can go as high as 4.0 if DNA <100 bp) For example, if you are using the large gel tray (250 mls of TAE) and your DNA fragments are between 200 - 1,000 bp, you should make a 1.0% agarose gel, which means you would add 2.5g of Agarose to 250 ml of TAE. Remeber, % (w/v) means grams/100 mls. So a 20% w/v would mean 20 g/100 mls. 4) Microwave the TAE/agarose solution long enough so that all the agarose is dissolved. For 500 mls this usually takes 6 minutes. For 250 mls it takes about 4 minutes. 5) Cool the TAE/agarose solution enough so that you can place your wrist on the Erlenmeyer and not scream in pain. 6) Add Ethidium Bromide (EtBr) to the gel for a final concentration of 0.5 µg/ml. EtBr is normally purchased in solution at 10 mg/ml. Add 5 µl to 100 ml of TAE/agarose solution. Scale accordingly to the amount of TAE/agarose solution you are preparing. 7) Pour TAE/agarose solution into gel tray. Leave undisturbed until gel hardens. 8) Gently remove end-pieces and well combs from gel 9) Place gel tray in gel box and fill gel box with TAE so that it barely covers the gel. Remember to place the gel so that the black lead is at the top of the gel and the red lead at the bottom. DNA is negatively charged (black) and will move through the gel towards the positive lead (red). 10) Optional: If PCR product does not contain a tracking dye (sucrose red or other) add tracking dye to each tube of PCR product. Generally use 5X stock of sucrose red, so add 5 µl 5X sucrose red to a 25 µl PCR reaction. 11) Carefully pipette 5 µl of DNA ladder into the first well of each row of wells. 12) Carefully pipette PCR sample into wells. Small well combs will hold up to 18 µl of sample, while larger combs will hold up to 50 µl of sample. 13) Place cover on Gel tray and turn on power supply. Generally run medium and large gels at 150 volts and small gels at 100 volts. Check to make sure electricity is flowing. Run gel until the red stripe of the tracking solution is about halfway between the rows of wells. 14) Turn off power supply. 15) Place gel on UV box and take picture. 16) Tape picture to PCR excel spreadhseet printout and label each lane on the gel. TAE buffer 726.6g TRIS Base 300 µl 0.5M EDTA pH 8.0 171.3 ml glacial acetic acid up to 3 L ddH2O Sucrose Red tracking dye (5X) 60g Sucrose 100 ml RNase free H2O Stir until dissolved 40 mg Cresol Red Stir until dissolved. Store at -20ºC

Hazard Identification and Risk of Exposure to the Hazards: Exposure to ethidium bromide in agarose gels

Exposure Controls Specific to Above Risk of Exposure: Always wear PPE: Lab coat, safety goggles, gloves. Dispose of ethidium bromide as indicated below.

Waste Generated and Disposal Methods: Liquid waste: Ethidium bromide must be disposed of in a hazardous waste container and stored in secondary containment until picked up by DEHS for hazardous waste disposal. Solid waste: Ethidium bromide contaminated tips should be collected in a waste container until picked up by DEHS for hazardous waste disposal. The amount of Ethidium bromide used in this protocol in the agarose gel is low enough that DEHS has concluded the gel can be discarded in the trash. Sharps containers will be sealed when ¾ full and placed in designated waste area. Refer to the Biological Waste Disposal procedures posted on the Tissue Culture room door for more information

Spill Response Procedures: Cover any spills with paper towels, and place towels in bucket labeled as hazardous waste. If exposed to hazardous chemicals, rinse with copious amounts of water and seek medical attention at Boynton Health Services, or Fairview Emergency room if after normal business hours.

Accident Response Procedures: If Incident Results in a Hazard Exposure ( i.e. face or eye splash, cut or puncture with sharps, contact with non-intact skin): • Encourage needle sticks and cuts to bleed, gently wash with soap and water for 5 minutes; flush splashes to the nose, mouth, or skin with water; and flush eyes at the nearest eyewash station with clean water for 15 minutes. • Call 911 or seek medical attention. - For urgent care employees may go to HealthPartners Occupational and Environmental Medicine (M/F day time or Urgent Care after hours), or UMMC- Fairview Hospital (24 hrs). You may seek medical attention at the closest available medical facility or your own healthcare provider. - Follow-up must be done by HealthPartners Occupational and Environmental Medicine. • Report the incident to your supervisor as soon as possible, fill out the appropriate documentation. - Employee First Report of Injury - Supervisor Incident Investigation Report • Send Incident Report Form to the IBC if exposure has occurred during work on an IBC protocol. • Report all biohazard exposures to the Office of Occupational Health and Safety (612- 626-5008) or [email protected]. Note: It is important to fill out all of the appropriate documents to be eligible to collect workers compensation should any complications from the hazardous exposure arise in the future.

Notes: (special record keeping such as inventories for toxins, reporting, training, etc. that may be required)

References: For further information view the UMN DEHS website containing Bio Basic Fact Sheets at http://www.dehs.umn.edu/bio_basicfacts.htm. For general information on Biosafety, access the Biosafety in Microbiological and Biomedical Laboratories (BMBL) 5th Edition from the CDC at BMBL http://www.cdc.gov/biosafety/publications/bmbl5/index.htm For Material Safety Data Sheets access the Public Health agency of Canada website MSDS http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php.