Haynes Lab:Notebook/Synthetic Biology and Bioinformatics for Predictable Control of Therapeutic Gene2/2013/01/07

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RNeasy Mini Kit

  1. Add 100μL of chloroform to each tube
  2. Shake vigorously for 20 sec and let sit for 2 min
  3. Centrifuge in cold room for 15 min @ 12,000G
  4. Pipet off clear phase from organic phase into new tube (warning: do not get pink stuff in the tube!)
  5. Add 70% ethanol into tube (1:1 ratio)
  6. Transfer to collection tube (we have K562 1, K562 2, SK-N-SH 1, and SK-N-SH 2)
  7. Spin tubes at top speed for 30 sec
  8. Discard collected liquid and add 700μL of RW1 Buffer
  9. Spin tubes at top speed for 30 sec
  10. Put collection vial in a fresh collector tube and discard of the old one
  11. Add 500μL of RBE Buffer
  12. Spin tubes at top speed for 30 sec then discard of the liquid waste
  13. Repeat the above two steps again
  14. Discard of the waste and spin again
  15. Transfer liquid to a fresh tube and add 30mL of RNase-Free H20
  16. Let tubes sit for 1 min
  17. Spin tubes for 1 min
  18. Store at -80°C