Monday, November 10, 2003

Description
Isogen RNA Extraction Protocol

Procedure
This protocol is for 200mg of leaf tissue
Add 0.25ml of ddH2O and 0.75ml of Isogen-LS to eppendorf tube (one per sample)

Vortex to mix

Hold mortar in liquid N2 to cool and place on alfoil

Add liquid N2 to mortar and pestle to freeze

Leave small spoon into N2 to keep cold

Place sample in mortar and homogenize in a circular motion, adding N2 as necessary to keep sample frozen

Once fully ground, use small spoon to transfer sample to eppendorf tube with Isogen-LS

Vortex on eppendorf shaking platform for 1 min

Incubate at 50C for 6 min

Mix samples on medium speed shaker for 5 min at rt

Add 200l chloroform to each tube and vortex to mix

Leave at room temperature for 2 to 3 minutes

Centrifuge for 15 minutes, 15000 rpm, 4C

Transfer upper clear layer (RNA) to fresh eppendorf tube (about 700 to 800l) DO NOT take any of the white protein phase dividing the two layers

Add 450l of isopropanol to RNA sample

Invert to mix

Leave for 5 to 10 minutes at room temperature

Centrifuge for 15 minutes, 15000 rpm, 4C, discard supernatant

Quick centrifuge at 4C, until reaches 15000 rpm then stop

Discard supernatant and add 1ml 70% EtOH

Centrifuge for 5 minutes, 15000 rpm, 4C, discard supernatant

Quick centrifuge at 4C, until reaches 15000 rpm then stop

Discard supernatant completely

Add 200l of ddH2O and vortex to resuspend RNA pellet. NOTE if pellet does not quickly resuspend, incubate in 60C waterbath

Store tube on ice

Phenol:Chloroform Purification

Add appropriate volume of chloroform to phenol to make a 1:1 solution

Add 200l of P:C to 200l RNA sample

Vortex for 3 minutes on vortex rack

Centrifuge for 5 minutes, 15000 rpm, 4C

Remove top layer (RNA) and transfer to fresh eppendorf tube

Add 200l of chloroform to solution in new tube

Vortex for 3 minutes on vortex rack

Centrifuge for 5 minutes, 15000 rpm, 4C

Remove top layer and transfer to fresh eppendorf tube

Add equal volume of 4M LiCl

Invert to mix

Incubate at -70C for 1 hr, or on ice in 4C cold room overnight

NEXT DAY

Centrifuge for 15 minutes, 15000 rpm, 4C, discard supernatant

Add 500l of 70 % ethanol and rotate tube to wash RNA pellet

Centrifuge for 5 minutes, 15000 rpm, 4C, discard supernatant

Quick centrifuge at 4C, until reaches 15000 rpm then stop

Discard supernatant completely

Add 100l of ddH2O and resuspend RNA pellet gently

Add 10l of 3M NaOAc and 250l of 100 % ethanol

Incubate at minuc 20C for 30 minutes, or at minus 80C (dry ice) for 15 minutes

Centrifuge for 15 minutes, 15000 rpm, 4C, discard supernatant

Add 500l of 70 % ethanol and rotate tube to wash RNA pellet

Centrifuge for 1 to 2 minutes, 15000 rpm, 4C, discard supernatant

Quick centrifuge at 4C, until reaches 15000 rpm then stop

Discard supernatant completely

Dry pellet in vacuum for 3 minutes

Add 20l of ddH2O and resuspend RNA pellet

Store at minus 30C overnight before proceeding to the next step

NEXT DAY

Quantify the RNA, determine volume required for 10g (one gel lane)

Store at minus 30C



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