5.1.1.2. RNA and cDNA preparation

  1. Centrifuge thawed aliquots for 2 min at 6,500 x g and 4°C.
  2. Resuspend bacterial pellets in TE buffer with lysozyme (15 mg/ml) and proteinase K (Qiagen).
  3. Vortex for 10 sec.
  4. Incubate 10 min at RT with constant shaking.
  5. Isolate RNA with RNeasy Plus Mini Kit and protocol “Purification of Total RNA for Animal Cells” (with on-column DNase I treatment).
  6. Elute RNA with 30 µl RNase-free water.
  7. Store at -80°C.
  8. Convert RNA to cDNA with RevertAid First Strand cDNA Synthesis Kit (Fermentas), using random hexamer primers.