Sequencing reservoir assembly

Introduction

This protocol details materials and methods to assemble Sequencing Reservoirs. The reservoir is composed of three major parts: reservoir Top, reservoir Bottom, and sealed in between the two, the sequencing substrate coated in immobilized surface primers.

A vacuum fixture is used to hold the reservoir bottom in place as the sequencing substrate is aligned on top of the gasket of the reservoir bottom. Once well aligned, the vacuum is turned on and will hold the slide firmly to the gasket. The gasketed surface of the reservoir top is then placed on top of the slide, and force is applied to lock the reservoir top legs into the reservoir bottom. Looking down into the reservoir through the top reveals about 20 mm² of the sequencing substrate. The top surface of the substrate is covered in immobilized surface primers and ready for sequencing library hybridization.

A press is used to compress the whole assembled sequencing reservoir to help ensure there are no leaks during subsequent steps.

Revision History

Materials and Equipment

Notes Before Beginning

Sequencing substrates and reservoir tops and bottoms should remain particle free. Components should be stored in clean air tight containers when not in use. For manipulation it is recommended to work in a clean room and/or within a laminar flow hood. All materials for reservoir assembly should be placed within clean space before starting procedure.

Procedure

1. Assemble Reservoir

1. Place reservoir bottom onto the vacuum block with the TPU gasket facing up and the elevated legs in the top left and bottom right corners
2. Using tweezers, carefully place the slide onto the bottom part of the reservoir with the oligo coated side facing up
3. Use tweezers to gently manipulate slide so it is centered over the reservoir bottom
4. Turn the vacuum on so the slide and bottom reservoir are help in place for next steps. Ensure slide is still centered over reservoir bottom
5. Screw on a cap to the reservoir top
6. Hold the reservoir top over the slide and reservoir bottom so the longer legs are positioned over the longer leg inserts of the reservoir bottom
   • Longer pieces of both parts should be at the top left and bottom right corners
7. Once properly positioned, carefully lower reservoir top onto reservoir bottom so the legs are resting on the leg inserts
8. Depress forcefully to finish assembling the reservoir
   • Listen for two audible “clicks” as the reservoir top is fitted into the reservoir bottom

2. Check assembly

1. Turn off vacuum holding reservoir in place and remove assembled reservoir
2. Inspect edges of glass slide to ensure it is still well centered between top and bottom pieces and there are no corners or sides protruding
3. Inspect gasket to slide contact of top and bottom pieces ensures there are no gaps between glass and gaskets

3. Press assembled reservoir to prevent leaking

1. Place reservoir cap-down in reservoir press fixture
2. Turn on press and wait 5 seconds
3. Turn off press and remove reservoir
4. Label reservoir for your experiment