This protocol is a robust method that will give you the best quality sequencing clean-up. It is more complex than a simple ethanol precipitation so should only be used when you are having clean-up difficulties. It was published as n-Butanol Purification of Dye Terminator Sequencing Reactions (1999).
1. Description of Sequencing Clean-up Process
Phenol extraction and butanol precipitation of sequencing reactions.
2. Chemicals involved in Phenol/Butanol Cleanup Process
- Phenol CAS# 108-95-2
- 1-butanol CAS#71-36-3
3. Potential Hazards
Inhalation of toxic vapors – phenol
Inhalation of harmful vapors – butanol
Skin contact with toxic and harmful liquids – phenol and butanol are harmful and readily absorbed through skin.
Skin burns – phenol burns skin
Fire hazard – butanol is flammable, phenol is combustible.
4. Personal Protective Equipment
Wear a lab coat, enclosed shoes, safety glasses or goggles and nitrile gloves for eye and skin protection.
5. Engineering/ Ventilation Controls
Use phenol and butanol in the fumehood.
6. Special Handling Procedures and Storage Requirements
Avoid using phenol and butanol near an open flame. Recap bottles immediately after use. Store source bottles of butanol in the flammables cupboard. Working stocks of solutions are to be kept in glass bottles and labeled correctly.
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 15min. Seek medical attention.
Eye exposure: Wash eyes for at least 15min, lifting the upper and lower eyelids. Seek medical attention immediately.
Small Spills: Notify others in the area of a spill. Do not attempt to clean up if you feel unsure of your ability to do so or if you perceive the risk to be greater than the normal laboratory operations. Cover spill with absorbent material (kitty litter) supplied in the Spill Kit. When absorbent is removed, wash contaminated area.
Large Spills: Notify others in the area of a spill. Turn off ignition sources in area. Evacuate area and post entrance ways to spill area. Restrict persons from the spill area until the clean up is complete. Remain in the area in a safe location to assist with response by emergency services.
8. Decontamination Procedures
After cleaning up spill with absorbent material, dispose of contaminated material in the appropriate solid waste container. Wash area of spill with water.
9. Waste Disposal Procedures
Disposal of the accumulated waste in the appropriate waste disposal container.
Phenol/Butanol Sequencing Cleanup Protocol
- Sequencing reactions that are 8ul volume in 0.2mL tubes
- MilliQ water
- 0.5ml tubes
1. Set up the appropriate number of 0.5ml tubes labeled as per the numbering system. Leave one set empty to be used in step 4. Add 300µl of butanol to the other set to be used in step 8.
2. To each sequencing reaction add 30µl of MilliQ water using the Eppendorf P50 multi channel pipette if cleaning a 96 well tray or a P200 if working with a smaller set of sequencing reactions.
3. Add 25µl of phenol to each tube using the Eppendorf P50 multi channel pipette if cleaning a 96 well tray or a P200 if working with a smaller set of reactions.
4. Transfer the samples to 0.5ml tubes using a P200 pippetor.
5. Vortex samples until cloudy. Do this by tilting the tube on its side and pulsing it against the vortex for 2 seconds twice. If the sample is still not cloudy at this point, repeat vortexing until it is cloudy.
6. Centrifuge in a table top centrifuge at 12000 rpm for 3min.
7. Remove the top layer (aqueous phase) to a fresh 0.5ml tube containing 300µl of butanol using a single channel P200. This step should be done immediately after the centrifuge has stopped and requires careful pipetting as you only want to take the top layer and not the white interface or the lower phenol layer. Try and take about 36µl of the top phase, but if there are white bits as you get closer to the interface, then leave them behind. If the sample is still cloudy after centrifugation then repeat the vortex step and then recentrifuge for 3min.
8. Vortex samples with butanol well. Do this by tilting the tube on its side and pulsing it against the vortex for 10 seconds twice. The butanol needs to be completely mixed in with the sample, if you see droplets at the bottom of the tube then vortex it until the sample and butanol are completely mixed.
9. Centrifuge in a table top centrifuge at 12,000 rpm for 10min. Position the tubes with the edge of the cap facing out, this will ensure you know the orientation of the DNA pellet.
10. Remove the supernatant from the sample. After the centrifugation step 10. The pellet is positioned in the bottom of the edge side of the tube. Remove the supernatant by briefly tilting the tube with the edge facing upwards. Do not jostle the tubes too much as you don’t want to disturb the pellet. Tilting the tube removes most of the supernatant, but to remove the remaining supernatant see step 12.
11. Briefly centrifuge the samples by holding the pulse of the centrifuge until it accelerates to max speed then stop the centrifuge. This is enough to bring down the remaining supernatant.
12. Remove the remaining supernatant from the bottom of the tube using an single channel P20. Position your pipette at the opposite corner of the bottom of the tube to the pellet. Do not disturb the pellet.
13. Place tubes in the 80 deg.C drying oven to remove any traces of butanol that are remaining. This should only take 5-10min.
14. Add 1µl (2µl if only loading half) of sequencing loading dye to the sample and vortex for 30s.
15. Store sample in the -20 deg.C.
It is a good ideal to aliquot out small lots of phenol in 0.5ml tubes and store them in the -20 deg.C freezer. Always take care when working with phenol!