Standard Operating Procedure for extraction of CandidaDNA from human samples
Title or Type of Procedure: Extracting DNA from human samples
P. I. Cheryl Gale Lab Location: 13-125 MCB
Original Issue Date:12/13/2010
Re-issue Date: 2/23/17
Prepared By: Tim Heisel
Procedural Methods and Materials: See following sheets.
Hazard Identification and Risk of Exposure to the Hazards: Candida albicanshas been classified as a Biosafety Level 2 (BSL2) organism. Hazards for BSL2 organisms include potential for aerosolization during routine manipulationn of Candidaspp. including transfer to test tubes and plates for growth, vortexing and/or centrifugation. Candidaspp. are not respiratory pathogens and extremely large numbers of organisms are required to cause any infection so the risks to laboratory personnel are minimal. Candidaspp. are normal yeast organisms in most human GI tracts and cause disease only in immunocompromised individuals. No food or drink is to be consumed in the laboratory. Quantities used are in the 0.1 - 2 µl range. Spill risk is negligible. Lab coats and gloves are worn at all times when handling yeast and yeast components (genomic DNA, protein). Any spills of yeast cultures are decontaminated with 10% bleach by volume.
Exposure Controls Specific to Above Risk of Exposure: Use of protective wear including gloves and laboratory coats is recommended. Screw cap tubes and centrifuge lids are generally used to minimize aerosolization. No sharp objects will be used to transfer live cultures (no Pasteur pipets, no needles, etc). Follow lab disposal plan for all materials used in this protocol and lab decontamination plan for any spill. NOTE: Any immunocompromised individuals present in the lab must wear a custom-fitted N95 or equivalent respirator when working with Candidaspp. to avoid potential infections. All personnel requiring N95 respirators will be enrolled in the OHS respiratory protection program.
Waste Generated and Disposal Methods: Follow Gale lab SOP for disposal of waste.
Spill and Accident Response Procedures: Follow Gale lab SOP for any spills or accidents.
Notes: All laboratory personnel must take Bloodborne and Other Pathogens Training.
Total DNA extraction from human-associated samples:
The yeast strains in the quantities used are non-hazardous. Gloves should be worn at all times. Standard procedure is to apply 10% bleach to all liquid cultures and autoclave all Petri-dishes and other plasticware that comes into contact with organisms for 60 min at 121° C. Make sure cultures are incubated with 10% final concentration bleach for at least 30 min. Wash any spills with 10% bleach, leaving in contact for at least 30 min., using gloves.
Centrifuge all samples in sealed microfuge tubes using a microfuge rotor with a sealed lid.
Transfer 250 mg of fecal sample into 2 ml microfuge tube containing the following:
2. Bead-beat samples for 60 seconds at 3,200 rpm and cool on ice for 10 minutes. Repeat two more times.
3. Centrifuge samples at 16,000 x g for 10 minutes.
4. Transfer aqueous (top) layer to fresh 2 ml microfuge tube. Dispose of pellet and phenol tube in the appropriately labeled hazardous waste container.
5. Add 0.7 x volumes of isopropanol and mix by inverting tubes.
6. Incubate at -20°C for 1 hour.
7. Centrifuge samples at 16,000 x g for 5 minutes. Discard supernatant in appropriately labeled hazardous waste container.
8. Allow DNA pellet to dry.
9. Resuspend DNA in 200 µl of TE buffer, pH 8.0
10.Add 200 µl 1:1 phenol:chloroform. Mix by inverting tubes.
11.Centrifuge samples at 16,000 x g for 10 minutes.
12.Transfer aqueous layer to fresh 1.5 ml microfuge tube. Dispose of phenol tube in the appropriately labeled hazardous waste container.
13.Add 1 ml of 100% ethanol. Mix by inverting tubes.
14.Incubate at -20°C for 1 hour.
15.Centrifuge at 16,000 x g for 5 minutes. Discard supernatant in appropriately labeled hazardous waste container.
16.Allow pellet to dry. Remaining procedure no longer has to be performed in chemical hood.
17.Resuspend pellet in 0.4 ml TE buffer, pH 7.5, plus 3 µl of 10 mg/ml RNase A.
18.Incubate at 37°C for 30 minutes, shaking every 10 minutes.
19.Add 10 µl 4 M ammonium acetate and 1 ml 100% ethanol. Mix by inverting tubes.
20.Incubate at –20°C for 30 minutes.
21.Centrifuge at 16,000 x g for 5 minutes. Discard supernatant in appropriately labeled hazardous waste container.
22.Allow pellet to dry.
23.Resuspend pellet in 50 µl TE buffer, pH 8.0.