LiCl RNA Extraction

1. Pipette 15 mL of RNA extraction buffer into a 50 mL falcon tube and add 200 μL β-mercaptoethanol (to a final concentration of 2%) and heat in water bath at 65°C.
2. Grind 1 g of plant tissue to powder in mortar and pestle with liquid N₂.
3. Transfer the powder to the preheated buffer vortex so it doesn’t clump and return the tube to waterbath at 65°C for 1 minute - then leave at room temperature while doing other samples.
4. Add 15 ml of chloroform isoamyl alcohol (CIAA) to the tube, vortex then add to centrifuge tube
5. Spin at 10,000 rpm on ss34 rotor for 10 mins at RT
6. Transfer top aqueous phase to a new centrifuge tube and again add equal volume of CIAA and spin again under same conditions
7. Transfer top aqueous phase to a falcon, estimate volume and add 0.25 volume of LiCl (10 M)
8. Incubate overnight in the fridge
9. Spin at 4°C 10,000 rpm ss34 for 20 mins pour off supernatant
10. Dissolve pellet in 500 μL of SSTE solution (preheated to 65°C) and transfer to ED tube
11. Add 500 μL of CIAA (500 μL) and vortex immediately add to eppendorf tube
12. Spin at 13,000 rpm for 10 minutes in bench top centrifuge
13. Put top phase into a new tube and add two volumes of -20°C ethanol to precipitate RNA (INVERT GENTLY 3-4 TIMES)
14. Put tube in -80°C for 30 mins
15. Pellet RNA at 13,000 rpm for 20 min at 4°C
16. Remove supernatant and dry pellet
17. Resuspend in RNase free water