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PDMS Chamber Fabrication

  • Version: v1.3
  • Last modified: 2026-08-18

What this is for

Nearly every wet step in the workflow -- hybridization, ligation, RCA, readout -- is run as a 100 μl reaction held over the tissue section. A punched PDMS sheet pressed onto the slide is what forms that well: it seals by conformal contact (no adhesive, no bonding step), holds the volume without spreading, tolerates 30--55°C incubations, and peels off cleanly between steps. This page covers casting the sheet and punching it into chambers.

Required Materials

  • PDMS: Sylgard 184 Silicone Elastomer Kit (Component A: base, Component B: curing agent)
  • Mold: 15 x 15 cm square mold
  • Punch: 5 mm diameter round microfluidic hole punch — see Punch the Chamber
  • Other: Weighing container, stirring rod

Equipment

  • Balance (0.1g precision or better)
  • Vibrator/mixer
  • Vacuum pump and desiccator (for degassing)
  • Oven (80°C capable)
  • Blade or cutting tool
  • Cutting mat / sacrificial backing board (to punch against)

Formulation

Weigh by mass, not volume — the two components have different densities and the ratio is defined by weight.

Component Parts 10:1 — standard 12:1 — stiffer
Sylgard 184 Component A (base) 10--12 109.1 g 110.8 g
Sylgard 184 Component B (curing agent) 1 10.9 g 9.2 g
Total per batch 120 g 120 g
  • Yield: 2 batches (240 g) fill 3 molds (15 x 15 cm)
  • Which ratio: use 10:1 for chambers — the softer material conforms to the slide and seals more reliably. 12:1 is harder, clearer and punches more cleanly, but is more brittle at the chamber rim.

Steps

1. Weigh

  • Tare the weighing container, then weigh Component A to the target in the table above.
  • Add Component B directly into the same container to the combined target (e.g. 109.1 g → 120.0 g for 10:1).
  • Component B is markedly more viscous — add it slowly and let it settle before reading the balance.

2. Mix

  • Pre-mix with stirring rod
  • Place on vibrator for thorough mixing: 5-10 min until uniform
  • Bubbles during mixing are normal

3. Degas

  • Transfer to vacuum desiccator
  • Apply vacuum for 15-30 min
  • Done when: no visible bubbles rising
  • Watch for overflow during degassing

4. Pour

  • Slowly pour degassed PDMS into mold
  • Pour from one side, let liquid self-level
  • Pop any remaining small bubbles with a needle
  • 2 batches (240g) = 3 molds

5. Cure

  • Place in oven at 80°C overnight (8-12 h)
  • Keep oven temperature stable; avoid opening door
  • Ensure mold is level

6. Demold & Cut

  • Cool to room temperature
  • Carefully peel cured PDMS from mold
  • Cut into 2.5 x 2.5 cm squares with a blade. This matches the 25 mm width of a standard microscope slide, so the block sits flush across the slide without overhanging either long edge.
  • Yield: a 15 x 15 cm mold gives 36 blocks (6 x 6); the 3-mold batch gives ~108.
  • Storage: Room temperature, long-term stable

7. Punch the Chamber

Goal: Remove the central cavity so the cut sheet becomes a well that holds the reaction volume over the section.

  • Lay the cut PDMS piece flat on a cutting mat, cured (smooth) side up.
  • Punch through completely with a single vertical press — do not twist and re-enter, which leaves a ragged wall that seals poorly and traps bubbles.
  • Confirm the plug releases cleanly; pick out any partially attached slug rather than tearing it, which nicks the chamber wall.
  • Deburr the rim if needed, then clean the sealing face with ethanol or isopropanol and let it dry before use.

Punch size

The standard chamber is punched with a 5 mm diameter round punch. Sold as a microfluidic hole punch (搜 "微流控打孔器") in a range of diameters — the lab's were bought on Taobao, so there is no catalogue number to quote; buy by diameter and shape, not by part number.

Punches are consumable: they blunt with use and a blunt punch tears rather than cuts, which is the usual cause of a leaking chamber. Keep a spare.

Chamber geometry -- why 5 mm x 5 mm

Footprint x thickness = the reaction volume, and every protocol on this site is written for 100 μl:

Value
Block footprint 2.5 x 2.5 cm (matches the 25 mm slide width)
Cast sheet thickness ~5 mm
Punch diameter 5 mm → 19.6 mm²
Nominal chamber volume ~98 μl

So the standard chamber holds the 100 μl reaction almost exactly, with no headroom to spare. Two practical consequences:

  • Do not overfill. At this geometry 100 μl fills the well to the brim; any more and it domes over the rim and wicks under the seal.
  • If you cast thinner, scale the reaction down rather than spreading it. A 3 mm sheet gives ~59 μl — run 50 μl reactions, do not try to fit 100 μl into it.

Seal quality

PDMS seals to clean glass by conformal contact alone. If reactions leak or dry out overnight, the usual causes are a dusty or wet slide surface, a torn chamber rim from twisting the punch, or a sheet cast too thin to apply even pressure. Reject chambers with visibly ragged walls -- they are the main source of evaporation losses in the 12--16 h RCA step.

Quality Check

  • Appearance: Transparent, colorless, smooth surface
  • Elasticity: Good elasticity, not easily torn
  • Bubbles: Minimal in surface and interior
  • Thickness: Uniform throughout

Troubleshooting

Problem Cause Solution
Sticky, doesn't set Inaccurate ratio or poor mixing Ensure precise weighing and thorough mixing
Too many bubbles Insufficient degassing Extend vacuum time or degas in stages
Uneven thickness Mold not level or poured too fast Level mold, pour slowly, gently tilt to spread
Incomplete curing Temperature or time insufficient Extend curing time or increase temp (max 100°C)
Ragged chamber wall Punch twisted or re-entered; blunt punch Single clean vertical press; replace the punch when it starts tearing rather than cutting
Chamber leaks / dries overnight Poor seal — dusty or wet slide, torn rim, sheet too thin Clean and dry both faces, discard torn chambers, cast thicker

Notes

  • Components A and B should be at room temperature before mixing
  • Mix in well-ventilated area
  • Wear gloves; avoid skin contact with uncured PDMS
  • Cured PDMS can be autoclaved (121°C, 20 min)
  • Clean cutting tools to avoid contamination
  • Clean PDMS surface with ethanol or isopropanol before use

Curing Reference

  • Room temperature: 24 h
  • 80°C: overnight
  • 100°C: 2 h
  • Standard ratio (10:1): moderate hardness and elasticity
  • Higher ratio (12:1): harder, more brittle, better transparency

For chamber use, the 10:1 standard ratio is preferred — the softer material conforms to the slide better and seals more reliably. 12:1 punches more cleanly but is more brittle at the rim.

Changelog

  • v1.3 — 2026-08-18 — Step list in the opening note follows RCA Protocol v6.0, which deleted the blocking step. MINOR — nothing about casting, curing or punching the chambers changed.

  • v1.2 — 2026-08-18 — Metadata block aligned to the house format: the Author and Purpose lines were removed, leaving Version / Last modified as on every other reagent page. Authorship is in git and the purpose is already stated in the frontmatter description and the opening admonition. No procedure, ratio or dimension changed. Format is now written down in docs/CONTRIBUTING.md.

  • v1.1 — 2026-07-29 — Reframed from a generic PDMS casting recipe to the chamber fabrication protocol it is used as: added why the chamber exists (holds the 100 μl on-slide reaction), a Punch the Chamber step (Section 7) using a 5 mm round microfluidic hole punch, seal-quality guidance, two new troubleshooting rows, and a note on ratio choice for chamber use. The A:B formulation is now a recipe table rather than prose. Chamber geometry recorded: 2.5 x 2.5 cm blocks cut from a ~5 mm sheet, punched 5 mm → ~98 μl, which is why the 100 μl reactions fill the well with no headroom; block size matches the 25 mm slide width.

  • v1.0 — 2025-10-30 — Initial PDMS casting protocol.

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