Reagent Protocols

Trizol Preparation

  1. Homogenize tissue with very cold mortar and pestle or homogenizer.
  2. Transfer tissue into 1.7mL Eppendorf tube or 25mL Falcon tube.
  3. Add 10µL TRIZOL / mg tissue, vortex thoroughly.
  4. Run tissue/TRIZOL mixture through a 20 gauge needle several times to complete homogenization.
  5. Let stand at room temperature for 5 minutes.
  6. Add 20µL chloroform / 100µL TRIZOL, vortex for 15 seconds, and leave at room temperature for 3 minutes.
  7. Centrifuge samples at maximum rpm for 15 minutes at 4°C.
  8. Transfer aqueous phase to a fresh tube (the use of Eppendorf PhaseLock Gel greatly decreases organic phase contamination at this step).
  9. Add 50µL isopropyl alcohol / 100µL TRIZOL and incubate at room temperature for 10 minutes.
  10. Centrifuge samples at maximum rpm for 10 minutes at 4°C.
  11. Remove supernatant.
  12. Wash RNA pellet with 80% ethanol and vortex.
  13. Centrifuge samples at 7,500g for 5 minutes at 4°C.
  14. Remove supernatant.
  15. Reconstitute pellet in 20µL of RNase free water.

Lysis Buffer

  • 50 mM Tris pH 8.0
  • 50 mM EDTA
  • 25 mM Sucrose
  • 100 mM NaCl
  • 1% SDS

Low TE pH 8.0

  • 10 mM Tris pH 8.0
  • 0.1 mM EDTA pH 8.0

RNAlater™

  • 25 mM Sodium Citrate
  • 10 mM EDTA
  • 70 g ammonium sulfate/100 ml solution, pH 5.2

(Information taken from patent information: US Patent 6,204,375)

Ribotyping (C.diff) Protocol

UAGC offers ribotyping via capillary electrophoresis. The primers and protocol we use is per the protocol described by
Indra A, Huhulescu S, Schneeweis M, Hasenberger P, Kernbichler S, Fiedler A, Wewalka G, Allerberger F, Kuijper E. J. Med. Microbiol. 57(11):1377-1382 doi:10.1099/jmm.0.47714-0
characterization_of_clostridium_difficile_isolates_using_capillary_electrophoresis-based_pcr_ribotyping.full_.pdf