How do I remove excess primer dimers?

  • Updated

If the final sample Tapestri library exhibits low-size off-target products (e.g., 200 – 240 bp) that make up more than 25 % (and less than 50 %) of all products based on molarity (number of molecules), then follow the steps outlined below.

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 After eluting 10 µL of Library PCR product after Ampure XP cleanup and using 1 µL for subsequent BioA assay, follow these steps:

  1. Add 16 µL of nuclease-free water to 9 µL sample for a total volume of 25 µL.

  2. Mix and quick-spin to collect the contents.

  3. Add 16.5 µL of Ampure XP reagent, at room temperature and well-mixed, to the above sample.

  4. Vortex for 5 seconds and quick-spin to collect the contents.

  5. Incubate the tube at room temperature for 5 minutes.

  6. Place on the magnet and wait 5 minutes for the beads to separate from the solution.

  7. Without removing the tube from the magnet, remove the clear liquid from the tube and discard.

  8. Add 100 μL of the freshly prepared 80 % ethanol, wait 30 seconds, and remove 100 μL of ethanol without disturbing the Ampure beads.

  9. Repeat Step 8 once for a total of two wash cycles.

  10. Remove all residual ethanol from the tube. Take the tube off the magnet and do a quick spin. Place the tube back on the magnet with the caps open and remove any residual ethanol.

  11. Dry the Ampure bead pellets in the tubes on the magnet by incubating at room temperature for 2 – 5 minutes. Avoid overdrying the beads.

  12. Remove the tube from the magnet. Add 9 μL of nuclease-free water into the tube. Vortex and quick-spin to collect the contents.

  13. Incubate the tubes at room temperature for 2 minutes.

  14. Place the tube onto the magnet and wait for at least 2 minutes or until the solutions are clear.

  15. Transfer 8 μL of purified PCR product from the tube to a new 0.2 mL PCR.

If the final sample Tapestri library exhibits low-size off-targets (e.g., 200 – 240 bp) that make up more than 50 % of all products based on molarity (number of molecules), contact Mission Bio Support at support@missionbio.com

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