Matt:LabNotes/2016-8-21

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SplintR Ligase Test

Reference

Experimental Outline

  1. Remove DNA from RNA (UHRR)
    • Shouldn't be completely necessary
  2. Biotinylate RNA
  3. Attach RNA to magnetic streptavidin beads
  4. V6 Padlock Probe capture to RNA

Remove DNA from RNA

  • Use Zymo DNA-Free RNA Kit
  • Sample: 4ul of UHRR (1.3 ug/ul)

DNase I Digest

Components Volume
UHRR (1.3ug/ul) 4
10X DNase I Buffer 5
DNase I 2
H2O 39
Total 50
  • Mix and incubate at 37C for 30min

Purify

  • Add 100ul RNA Binding Buffer and mix
  • Add 150ul 100% EtOH and mix
  • Transfer to Zymo-Spin IC Column in Collection Tube
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 400ul RNA Prep Buffer
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 800ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Add 400ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Centrifuge at 13,000rcf for 2min and discard flow through
  • Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min
    • Centrifuge at 10,000rcf for 30sec
  • Quantify with Nanodrop or Qubit

Biotinylate RNA

Attach RNA to Streptavidin Beads

  • MyOne C1 protocol
  • Make Solutions A & B and Binding&Wash Buffer
    • Solution A:
    • Solution B:
    • 2X B&W Buffer: 16ml 5uM NaCl + 80ul 5uM EDTA + 100ul 4uM Tris-HCl + 23.82ml H2O
  1. Resuspend beads by vortexing
  2. Transfer 100ul of beads (10ug/ul) to new tube
  3. Pull down by magnet 2min and remove supernatant
  4. Wash 3x with 100ul 1X B&W Buffer by resuspending and then removing supernatant
  5. Wash 2x with 100ul Solution A for 2 min each
  6. Wash 2x with 100ul Solution B
  7. Resuspend the beads in Solution B
  8. Add biotinylated RNA
  9. Incubate 15min at RT gently rotating
  10. Pull down with magnet for 3min
  11. Wash 3x with 1X B&W Buffer
  12. Resuspend in 1ml 1X PBS

V6 Padlock Probe Capture