Jun 25, 2026
DNA Sterilization Techniques Protocol 2026
- Katrina Lohan1,
- Ruth DiMaria1,
- Calli Wise1,
- Emma Palmer1,
- Tara Sill1,
- Lael Collins1
- 1Smithsonian Environmental Research Center, Coastal Disease Ecology Lab
- Coastal Disease Ecology Lab - SERC
Protocol Citation: Katrina Lohan, Ruth DiMaria, Calli Wise, Emma Palmer, Tara Sill, Lael Collins 2026. DNA Sterilization Techniques Protocol 2026. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbm7xnvpk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: June 25, 2026
Last Modified: June 25, 2026
Protocol Integer ID: 319833
Keywords: Sterilization, Bleach, Ethanol Flame Sterilization, Bleach Sterilization, DNA Sterilization, dna sterilization techniques protocol, flame sterilization, undesired dna, dissecting utensil, bleach, main method, protocol
Abstract
There are two main methods to destroy undesired DNA from dissecting utensils. Bleach wash or flame sterilization.
Guidelines
** New users must be trained by a technician or post-doc! **
Whenever in the lab, all individuals must wear shoes that completely cover their feet.
Whenever working with any chemicals, reagents, or DNA, all individuals must wear clean nitrile gloves and are responsible for understanding any hazards associated with those chemicals (Safety Data Sheet; SDS).
Wear gloves when working with bleach and work in a well-ventilated area.
This protocol was developed with the assumption that the end-user possesses baseline knowledge of appropriate aseptic/clean techniques and a comprehensive understanding of the potential risks of contamination inherent to the workflow.
** If there are any questions or concerns, please do not hesitate to ask Ruth DiMaria or Katrina Lohan **
When collecting tissue samples for DNA analysis, take steps to prevent cross-contamination due to DNA carrying over from one sample to the next, either by the utensils, your gloves, or the working surfaces (dishes, trays, etc.).
This is particularly important for molecular analyses examining symbionts, as the DNA of the symbionts is already in low quantity (compared to the DNA of the host) and the molecular assays are optimized to detect very low quantities of DNA.
Safety warnings
Chlorine bleach destroys DNA. Therefore, when using this sterilization method, you want to take steps to prevent any bleach from contacting, and potentially destroying, your samples.
Chlorine bleach is routinely used to sterilize surfaces and equipment. Bleach is an oxidizer and corrosive. The active ingredient, sodium hypochlorite, is incompatible with several reagents and chemicals used in the laboratory and poses safety risks. Mixing with guanidine salts (e.g., guanidine hydrochloride) will create chlorine gas. Mixing with alcohols (e.g., ethanol, isopropanol) will create chloroform. For more detailed information, refer to the Sodium Hypochlorite (Bleach) Safety Fact Sheet. Bleach can react negatively with other chemicals, including reagents used in DNA extraction (e.g., guanidine hydrochloride creating noxious chloride gas). Take precautions when using bleach in workspaces. Refer to SDS documents and read safety precautions carefully.
Wear gloves when working with bleach and work in a well-ventilated area. Lab coat and safety eyewear personal protective equipment (PPE) are available.
For flame sterilization:
- Only work in designated spaces for hot work with adequate ventilation. Verify the Hot Work Permit for the space is current before starting.
- Denatured ethanol includes additives that can be irritating when burned. Keep this in mind when using the flame sterilization method and work in a well-ventilated space. The CDE lab does not have denatured ethanol as the additives interfere with downstream genetic workflows.
- Ethanol is a flammable liquid used to sterilize metal utensils. As a precaution, use an extra Pyrex dish to cover the beaker of ethanol if it is accidently ignited by heated utensils.
- Participants must be knowledgeable of the location of the nearest fire extinguisher, manual pull station, phone, and exits.
- Check that the hot work equipment (burner, lighter) is in good condition before starting.
- Make sure to remove all combustible materials before starting. Do not wear loose clothing and pull back hair.
Bleach Wash
To be an effective disinfectant, diluted bleach solutions must contain between 0.5% - 2% sodium hypochlorite. If the stock bleach bottle contains 5.25% sodium hypochlorite (generally most household bleach is around this percentage) then a 10% dilution of the stock will produce a final 0.53% hypochlorite solution which is the minimum amount needed to be an effective disinfecting agent.
The water rinse step is meant to help remove residual bleach. If you are processing a large volume of samples, the water rinse in the second beaker will begin to accumulate bleach and essentially become a second diluted bleach rinse. Therefore, you will want to change/refresh the water in the second beaker at least 1x when processing a large number of samples (e.g., every 50 samples).
If using a large number of utensils (e.g., batch sample processing), you can put them into a bleach bath as you work and then rinse/dry simultaneously at the end. Do not let utensils sit in the bleach bath or water for an extended time otherwise they risk damage from corrosion and rusting.
Bleach baths (10%) must be made fresh each day to be effective at sterilizing equipment. Bleach combined with water breaks down quickly into salt and water within 24 hours; after which point it is no longer effective as a sterilizing agent. When handling bleach work in a well-ventilated area and wear nitrile gloves. Protective eyewear and lab coats are available. Do not leave items in the bleach bath for longer than 30 minutes. Bleach (even diluted 10%) is corrosive and will degrade plastics with prolonged exposure.
Wipe any excess tissues off the utensil with a clean paper towel.
Dip utensil in a diluted bleach solution (10-15%) for 5-10 seconds. Remove and shake off excess liquid.
Rinse under running freshwater or swirl in a water bath to remove residual bleach. Can use tap water, D.I. water is preferred if available.
With fresh paper towels, dry utensil as much as possible before proceeding. Once dried, no bleach remains, so ensuring the utensils are dry is one way to ensure that no bleach gets into your sample.
Once sterilized, do not place utensils onto a non-sterile surface or they will need to be re-sterilized.
Change out water bath every 30-50 samples.
Flame
Only use dissecting instruments that are metal and can be exposed to high heat and flame. Plastic dissecting instruments will melt.
Wipe any excess tissues off the utensil with a clean paper towel.
Dip the working end of the utensil into a beaker of 95% ethanol.
Ignite the working end and let the ethanol burn off (helps to place utensil in a Pyrex dish while the ethanol burns off). Be mindful of your hand placement in relation to the ethanol-soaked area before ignition.
Wait until the utensils are cool (~15 seconds) before proceeding to the next sample.
Once sterilized, do not place utensils onto a non-sterile surface or they will need to be re-sterilized.
Negative Control
If concerned about potential DNA contaminants in the surrounding sampling environment, collect a negative control sample.
Place an empty tube (e.g., 1.5mL microcentrifuge tube; same tube being used for samples) on the countertop where you are processing samples for genetics with the lid open.
When you are finished collecting samples, close the lid to the tube.
Label as "Atmospheric Control" with the collection date (MM/DD/YYYY). It will be processed in the same manner as the other genetic samples.
Repeat for each sampling event (assuming sampling events are occurring on different days).