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