Sequencing substrate preparation

Substrate preparation

Introduction

This protocol details materials and methods used to clean and activate quartz substrates for Sequencing Reservoirs.

The quartz substrates go through three stages of cleaning prior to activation: solvent, alkaline, and then an acid clean. Next, the clean slides are activated with piranha acid.

Commercially available MCP4 polymer is deposited on the surface of the activated substrate to provide a three dimensional layer of active NHS groups for oligonucleotide attachment.

Oligonucleotides with 5’ amine modifications are coupled to the slide surface. After blocking, washing, and drying the slides, they are ready to be assembled into Sequencing Reservoirs.

Assembled sequencing Reservoir with forward and reverse surface primers immobilized to sequencing substrate
Created with BioRender.com and used with permission

Revision History

Document Version Number Date Description of Change
Sequencing substrate preparation V1.0 01/2024 Original document -KF

Materials and Equipment

Material or Reagent Supplier Order Information
Polished 10±0.1 x 10±0.1 x 0.5 mm Z-Cut Quartz UQG Optics custom part
Semiconductor grade 99.5% Isopropanol Alcohol VWR BDH2027
Semiconductor grade acetone VWR BDH2025
Semiconductor grade methanol VWR BDH2029
Reagent alcohol VWR BDH1156
deionized or 18.2MΩ water various, in-house system ideal n/a
Potassium Hydroxide Spectrum Chemical P1325
Hydrochloric acid (1N) Sigma Aldrich 1506961000
Sulfuric acid (5M) Sigma Aldrich 1.60315.1000
Hydrogen peroxide (30%) BeanTown Chemical 219920
MCP4 Kit: 5x Coat on Coating Solution, MCP4 Polymer Concentrate, Spot on Spotting Solution, 2x Block on Blocking Solution Lucident Polymers MCP4 Kit
2x SSC Buffer with 0.1% SDS various n/a
Surface forward primer: /5AmMC12/CAAGCAGAAGACGGCATACGA*G*A*T IDT Standard desalted lyophilized DNA oligo; 100 nmole minimum scale; /5AmMC12/ = 5’ Amino modifier C12; * indicates phosphorothioated DNA bases
Surface reverse: /5AmMC12/UAAUGAUACGGCGACCACCGAGAUCTA*C*A*C IDT Standard desalted lyophilized DNA oligo; 100 nmole minimum scale; /5AmMC12/ = 5’ Amino modifier C12; * indicates phosphorothioated DNA bases
nuclease free water various n/a
DNA microarray printer reagents various n/a
Equipment Supplier Order Information
Vertical Slide Holder 454 Bio Open Source Download
Air tight container for slide holder various n/a
Teflon Coated Forceps various n/a
At least 3 250 mL Beakers various n/a
Sonicator various n/a
Vertical slide holder diH2O shower fixture 454 Bio Open Source CAD
Slide cassette diH2O shower fixture 454 Bio Open Source CAD
Slide Coating Cassette Assembly 454 Bio Open Source CAD
Filtered Compressed Nitrogen, adjustable pressure various n/a
Slide drying fixture 454 Bio Open Source CAD
80º C Vacuum oven various n/a
Vertical slide reaction chamber 454 Bio Open Source CAD
50º C oven various n/a
DNA microarray printer various n/a

Notes Before Beginning

Procedure

1. Clean Z-Cut Quartz Slides

  1. Solvent clean
    1. Submerge slides vertically in a bath of semiconductor grade acetone
      • Sonicate 5 minutes
    2. Transfer slides to a bath of semiconductor grade isopropyl alcohol
      • Sonicate 5 minutes
    3. Transfer slides to a bath of methanol
      • Sonicate 5 minutes
  2. Alkaline clean
    1. Transfer slides to a bath of 10% potassium hydroxide in methanol (% w/v)
      • Sonicate 20 minutes at 50º C
    2. Transfer slides to a bath of reagent alcohol
      • Sonicate for 20 minutes at 50º C
    3. Repeat steps 1.2.1 through 1.2.2 for a total of two potassium hydroxide and two reagent alcohol washes
    4. Transfer slides to a water bath
      • Sonicate for 5 minutes
    5. Transfer slides to a new water bath
    6. Repeat steps 1.2.4 through 1.2.5 for a total of 3 washes with sonication
    7. Transfer slides to a methanol bath
      • Sonicate for 5 minutes
  3. Acid clean
    1. Transfer slides into a fresh bath containing a solution of 50% hydrochloric acid in methanol
      • Sonicate for 30 minutes at 60º C
    2. Transfer slides to a water bath
      • Sonicate for 5 min.
    3. Repeat step 1.3.2 two more times for a total of three washes, using a fresh water bath for each wash
    4. Dry slides thoroughly with filtered compressed nitrogen
      • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface
    5. Store until ready to proceed to acid cleaning step

2. Piranha activate clean slides

  1. Transfer slides to a bath containing piranha acid
    • Prepare piranha acid by combining 1 volume 30% hydrogen peroxide to 3 volumes of 5 M sulfuric acid
    • Sonicate for 60 min at 60º C.
  2. Transfer slides into a water bath
    • Sonicate for 5 min.
  3. Repeat step 2.2 four times for a total of 5 water washes with sonication, using a fresh water bath each time
    • Ensure all traces of piranha acid are removed
  4. Dry slides thoroughly with filtered compressed nitrogen
    • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface

3. MCP4 polymer deposition

  1. Prepare MCP4 working solution by combining the following reagents:
    • Solution Final Concentration Volume for 1 mL
      Nuclease free water 780 – 796 µL
      5x Coat On Coating Solution 1x 200 µL
      50x MCP4 Polymer Concentrate 0.2 – 1x Determine ideal concentration experimentally 4 – 20 µL
    • Note: DO NOT add polymer to solution until slides are ready for incubation
  2. Incubate activated slides in MCP4 Working Solution for 30 minutes
    • Slides can be submerged or assembled in fixtures that allow for a pool of working solution to cover a desired area
  3. After incubation, slides need to be aggressively washed in deionized water
    • Submerge in running 5 minutes, or submerge in multiple passive baths of deionized water. If slides are assembled in fixtures, recommend disassembling and washing an addition 1-3 minutes vertically in running water.
  4. Dry slides thoroughly with filtered compressed nitrogen
    • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface
  5. Cure dry slides at 80º C under high vacuum

4. Oligo immobilization

  1. Prepare a 250 uM each solution of forward and reverse surface primers in nuclease free water
    • Concentrations can be optimized experimentally
  2. Determine ideal spotting conditions for your DNA microarray printer
  3. Surface primer solution can be diluted 1:1 with Spot on Spotting Solution when ready to spot primers onto surface
  4. Incubate slides overnight

5. Blocking, washing, drying

  1. Prepare 1x Block on Blocking solution by diluting stock 1:1 in nuclease free water
  2. Fill vertical reaction chambers with 270-300 uL of solution
  3. Transfer slides from overnight incubation into the pre-filled reaction chamber
  4. Incubate at 50 C for 30 minutes
  5. Place slides vertically in a deionized water bath
    1. Incubate 5 min in running water
  6. Transfer slides to a 2x SSC + 0.1% SDS solution and incubate 10 minutes with enough shaking to agitate the solution without dislodging slides
  7. Place slides vertically in a deionized water bath
    1. Incubate 5 min in running water
  8. Dry slides thoroughly with filtered compressed nitrogen
    • Any residual moisture will dry onto slide, TIRF system is very sensitive to any residue, debris, or dried material on slide surface