One pot sequencing buffer

Sequencing buffer


This protocol describes the procedure to prepare one pot sequencing buffer.

Revision History

Document Version Number Date Description of Change
One pot Buffer V1.0 Jan 2024 original JW

Materials and Equipment

Item Vendor Cat. No
ThermoPol Buffer, 10x NEB B9004S
MgSO₄, 100 mM NEB B1003S
TMAC (tetramethylammonium chloride) Sigma T3411
DTT (Dithiothreitol) Sigma 43815
PEG 8000, 50% (w/v) RIGAKU 1008054
NaOH, 1 M Sigma 79724
Water, Nuclease free IDT 11-05-01-04
Whatman anotop Syringe filter, 0.02 µm Cytiva 6809-1002
Pipette and tips (1000 µL, 200 µL, 10 µL) varous n/a
Pipette controller and serological pipettes various n/a
pH meter various n/a


  1. Open the sequencing buffer worksheet.
  2. Enter the required information.
  3. Bring 10X ThermoPol buffer, 100 mM MgSO₄ and DTT from the -20°C freezer to room temperature for 30 minutes to thaw
  4. Bring all other chemicals to a clean area.
  5. Use a clean container (for example, a glass beaker) to prepare the buffer.
  6. Add the required volume of each liquid component to the container.
  7. Record the lot information and the quantity added to the worksheet.
  8. Make sure the DTT bottle reaches room temperature and there is no condensation on the bottle before opening the cap.
  9. Return DTT to the freezer once finished.
  10. After all chemicals are added, fill the container with the nuclease free water to 1 mL less than the total volume.
  11. Stir slowly to mix.
  12. The pH should be around 8.3-8.4. Adjust pH to 8.8 at 25°C with 1 M NaOH.
  13. Add nuclease free water to bring mixture up to total volume.
  14. Filter the buffer with 0.2 µm filter.
  15. Aliquot 1.5 mL buffer to 2.0 mL clean centrifuge tubes for storage at -20°