4.3.3. RNA lysis/stabilization buffer

  1. Fill a 1l beaker with 300 ml of nuclease-free water and insert a large magnetic stir bar.
  2. Following safety procedures (http://www.sciencelab.com/msds.php?msdsId=9927539 ) add:
    94.53g guanidine thiocyanate (CH5N3·CHNS; MW = 118.16) (Sigma #50981),
    30.45g ammonium thiocyanate (CH4N2S; MW = 76.12) (Sigma #43135),
    33.4ml of 3M sodium acetate (NaOAc), pH 5.5 ml ultrapure molecular biology-grade (USB # 75897 or Sigma #71196).
  3. Stir until completely dissolved.
  4. Pour into 1l graduated cylinder and bring up to 550ml with nuclease-free water.
  5. Pour from graduated cylinder into autoclave-safe desired 1l bottle.
  6. Add: 50 ml glycerol (C3H8O3; MW=92.09 g/mol) (Sigma # G6279) and 20 ml Triton-X 100 (Sigma #T8787).
  7. Autoclave on liquid cycle for 15 min with slow exhaust.
  8. Remove from autoclave immediately, cool and store at 4oC.

This makes a total volume of 620ml.