PCR: Cycling on a Microscope Slide

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PCR: Cycling on a Microscope Slide


Question:

What are some suggestions for performing PCR on a microscope slide?


Answer:

For PCR (or any enzymatic reaction) performed on slides, here are some suggestions:

  • Add 0.05–0.1% BSA or other carrier protein to the reaction mix in order to prevent DNA polymerases, DNA template, or other reaction components from binding to the glass surface
  • Make sure the tube reaction is truly optimized, especially for the annealing temperature
  • Starting with the protocol for the optimized tube reaction, try a series of optimization steps (especially for the annealing temperature) in the slide format without tissue present to verify that the reaction conditions work on the slide
  • When tissue is first used, try optimizing the protease digestion steps in the sample protocol. We recommend that you optimize protease digestion, probe hybridization, and color detection before adding the amplification steps
  • Optimize the protease concentration and digestion time for each new tissue type and each new lot of protease
  • When performing the amplification steps, minimize the total number of cycles performed (try only 15–20 cycles) and minimize the total time spent above 90°C to prevent tissue damage

We suggest you use Frame-Seal™ in situ PCR and Hybridization Slide Chambers. These are easy-to-use, strongly adhesive chambers with flexible plastic coverslips. They provide vapor-tight sealing for applications such as FISH, polony, in-situ PCR, and PRINS, and allow samples to be recovered easily. The seal withstands temperatures up to 97°C. Frame-Seal™ in situ PCR and Hybridization Slide Chambers are available in a range of sizes; 9 x 9 mm, 25 μl (SLF0201), 15 x 15 mm, 65 μl (SLF0601), 17 x 28 mm, 125 μl (SLF1201) and 19 x 60 mm, 300 μl (SLF3001)

Note: Please see the introduction to the Frame-Seal Incubation Chambers for Sealing Reactions on Slides instruction manual, Bulletin 03526.


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