One pot sequencing buffer

Introduction

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

Procedure

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°