Linear DISCS preparation

DISCS Preparation

Introduction

454’s One Pot Sequencing relies on the 365nm UV TIRF to deblock Lightning Terminator dye linked terminator groups. Stray UV light also deblocks the free Lightning Terminators™ in the bulk solution. The resulting deprotected dNTP competes with LTs during incorporation by Therminator and causes significant leading.

We have developed DISCS (Dark Base In-Situ Cleanup System): which includes Bst 3.0 DNA polymerase from NEB and a set of duplex oligos to quickly consume the deblocked dNTP (dark bases). DISCS is crucial for the success of One Pot Sequencing.

This protocol describes the procedure to prepare linear DISCS for one pot sequencing.

Revision History

Document Version Number Date Description of Change
DISCS V1.0 Jan 2024 original JW

Materials and Equipment

Item Vendor Cat. No
ThermoPol Buffer, 10x NEB B9004S
IDTE, pH 8.0 IDT 11-05-01-09
A_DISC_F IDT AAAAAACTATGACCGTGATTAGGCCAAGCTCGCACG
A_DISC_R IDT AAAAAACGTGCGAGCTTGGCCTAATCACGGTCATAG
C_DISC_F IDT AAACCCCTATGACCGTGATTAGGCCAAGCTCGCACG
C_DISC_R IDT AAACCCCGTGCGAGCTTGGCCTAATCACGGTCATAG
G_DISC_F IDT AAAGGGCTATGACCGTGATTAGGCCAAGCTCGCACG
G_DISC_R IDT AAAGGGCGTGCGAGCTTGGCCTAATCACGGTCATAG
T_DISC_F IDT AAATTTCTATGACCGTGATTAGGCCAAGCTCGCACG
T_DISC_R IDT AAATTTCGTGCGAGCTTGGCCTAATCACGGTCATAG
Water, nuclease free IDT 11-05-01-04
Thermal cycler various n/a
Pipettes and tips (200 uL, 20 uL, 2 uL) various n/a

Procedure

  1. Resuspend fresh oligo pellets from IDT with IDTE to 200 uM stock concentration.
  2. Open the linear DISCS worksheet.
  3. Enter the required information and the intended volume for each DISCS.
  4. Prepare four DISCS individually by mixing 10x ThermoPol buffer, forward oligo, reserve oligo and nuclease free water following the worksheet.
  5. Aliquot the mix to 200 uL PCR strip tubes with no more than 50 uL into each tube.
  6. Anneal forward and reverse oligos together by a step-down annealing protocol:
    1. 90°C for 60 seconds.
    2. 75°C for 30 seconds.
    3. 70°C for 30 seconds.
    4. 60°C for 30 seconds.
    5. 50°C for 30 seconds.
    6. 40°C for 30 seconds.
    7. Store at 10°
  7. Mix all solutions together equally and mix well.
  8. Aliquot 20 uL to each tube and store at -20°