total RNA isolation from plants

starting material: 2 ml eppendorf cup filled to the 500 ml mark with frozen and grinded plant material

• add 1 ml GTC-lysis buffer + 1% b-Me
• homogenise with pistill
• shake for 30 min at 37 °C
• spin 15 min at 4 °C
• put supernatant in new 2 ml eppendorf
• add 1/50 volume 3M NaAcetate and 0.75 volume ethanol
• incubate 2 h - overnight at -20 °C
• spin 15 min at 4 °C, remove supernatant
• resuspend pellet in 700 ml TES
• add 700 ml phenol, shake for 5 min
• centrifuge for 5 min
• take 650 ml upper phase into a new 2 ml eppie
• add 700 ml chloroform, shake for 5 min
• centrifuge for 5 min
• take 600 ml upper phase into a new 2 ml eppie
• add 200 ml 8M LiCl
• incubate overnight at 4 °C
• spin 15 min at 4 degrees, remove supernatant
• redissolve pellet in 300 ml water
• add 25 ml 3M NaAcetate and 750 ml ethanol
• incubate 2h - overnight at -20 °C
• spin 15 min at 4 °C
• remove supernatant
• redissolve pellet in 30 - 50 ml water

GTC-lysis buffer

TES