The last stage in preparing a DNA fragment for sanger sequencing is resuspending the cleaned sequencing products for injection onto the DNA sequencer. Traditionally pure water is used to resuspend these DNA products, but pure water exposed to the atmosphere absorbs CO2 to become a weak carbolic acid solution (pH of around 5.5). When the DNA products are stored at room temperature on the instrument for long periods of time (6 or more hours) the sequencing fragments can degrade in this low pH environment. In addition, water provides no protections from any DNases that might have been introduced to the samples.
The use of a low concentration Tris EDTA solution can help avoid both these problems. The Tris helps keep the sequencing fragments at an alkaline pH and the EDTA can helps inactivate DNases. In addition, the use of this solution helps normalize the DNA fragment size by competing with the small DNA fragment, thus biasing the overall injection profile towards larger fragments and helping to avoid overloading the instrument detector.
1. Description of Tris/EDTA Resuspension Buffer
- 10mM Tris / 0.1 mM EDTA
2. Chemicals used in the Tris/EDTA Resuspension Buffer
- Tris base (CAS Number 77-86-1)
- EDTA (CAS Number 60-00-4 )
- Molecular grade water
3. Potential Hazards
- Low hazard risk from chemicals as they are in relatively low concentrations and microlitre volumes.
4. Personal Protective Equipment
- Wear a lab coat, enclosed shoes, safety glasses or goggles and latex or nitrile gloves for eye and skin protection.
5. Engineering or Ventilation Controls
6. Special Handling Procedures and Storage Requirements
7. Spill and Accident Procedures
- Skin Exposure: rinse the affected skin with plenty of water while removing contaminated clothes and shoes. Rinse for at least 15 min. Seek medical attention if needed.
- Eye exposure: Wash eyes for at least 15min, lifting the upper and lower eyelids. Seek medical attention if needed.
- Dry Burn: rinse the affected skin with plenty of water. Rinse for at least 15 min. Seek medical attention if needed.
8. Decontamination Procedures
- Wipe up any spills with paper towels and water. Dispose of contaminated waste in the clinical waste container.
9. Waste Disposal Procedures
- Disposal of the accumulated waste in the appropriate waste disposal container.
- Prepare a stock solution of 1M Tris base / 10 mM EDTA from powder in molecular grade water and sterilise. Do not pH the stock solution.
- Prepare a series of 5 mL aliquotes of molecular grade water. Sterilise by autoclaving to remove dissolved CO2.
- Directly before use dilute the stock Tris/EDTA solution to the working concentration of 10mM Tris/0.1 mM EDTA (1:100) dilution by adding 50 µL of stock solution to a 5 mL aliquot of sterile water and mix by gentle inversion. Do not vortex.
- Add 10 µL of the 10/0.1 Tris EDTA solution to each tray well containing the cleaned DNA sequencing products and resuspend using gentle pipetting or vortexing. Cover with plastic seal.
- Load onto the DNA sequencer as per usual.
- Make the working solution fresh each day from the stock buffer. This will prevent the working solution absorbing CO2 from the atmosphere and/or acquiring bacterial contamination which can introduce DNases.
- Avoid adding small cations to the solution by not adjusting the pH as these compete with the DNA for loading onto the sequencer capillary. The exact pH of the stock and working solution is not critical provided that the pH is above 7.
- It is a good idea to make a batch of 5 mL sterile water solutions and autoclave them. This way you can make up a fresh working solution each day in a few seconds.
- Discard any leftover working buffer at the end of the day.