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Isogen RNA Extraction Protocol
Posted on 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 50°C for 6 min
Mix samples on medium speed shaker for 5 min at rt
Add 200µl chloroform to each tube and vortex to mix
Leave at room temperature for 2 to 3 minutes
Centrifuge for 15 minutes, 15000 rpm, 4°C
Transfer upper clear layer (RNA) to fresh eppendorf tube (about 700 to 800µl) – DO NOT take any of the white protein phase dividing the two layers
Add 450µl of isopropanol to RNA sample
Invert to mix
Leave for 5 to 10 minutes at room temperature
Centrifuge for 15 minutes, 15000 rpm, 4°C, discard supernatant
Quick centrifuge at 4°C, until reaches 15000 rpm then stop
Discard supernatant and add 1ml 70% EtOH
Centrifuge for 5 minutes, 15000 rpm, 4°C, discard supernatant
Quick centrifuge at 4°C, until reaches 15000 rpm then stop
Discard supernatant completely
Add 200µl of ddH2O and vortex to resuspend RNA pellet. NOTE if pellet does not quickly resuspend, incubate in 60°C waterbath
Store tube on ice
Phenol:Chloroform Purification
Add appropriate volume of chloroform to phenol to make a 1:1 solution
Add 200µl of P:C to 200µl RNA sample
Vortex for 3 minutes on vortex rack
Centrifuge for 5 minutes, 15000 rpm, 4°C
Remove top layer (RNA) and transfer to fresh eppendorf tube
Add 200µl of chloroform to solution in new tube
Vortex for 3 minutes on vortex rack
Centrifuge for 5 minutes, 15000 rpm, 4°C
Remove top layer and transfer to fresh eppendorf tube
Add equal volume of 4M LiCl
Invert to mix
Incubate at -70°C for 1 hr, or on ice in 4°C cold room overnight
NEXT DAY
Centrifuge for 15 minutes, 15000 rpm, 4°C, discard supernatant
Add 500µl of 70 % ethanol and rotate tube to wash RNA pellet
Centrifuge for 5 minutes, 15000 rpm, 4°C, discard supernatant
Quick centrifuge at 4°C, until reaches 15000 rpm then stop
Discard supernatant completely
Add 100µl of ddH2O and resuspend RNA pellet gently
Add 10µl of 3M NaOAc and 250µl of 100 % ethanol
Incubate at minuc 20°C for 30 minutes, or at minus 80°C (dry ice) for 15 minutes
Centrifuge for 15 minutes, 15000 rpm, 4°C, discard supernatant
Add 500µl of 70 % ethanol and rotate tube to wash RNA pellet
Centrifuge for 1 to 2 minutes, 15000 rpm, 4°C, discard supernatant
Quick centrifuge at 4°C, until reaches 15000 rpm then stop
Discard supernatant completely
Dry pellet in vacuum for 3 minutes
Add 20µl of ddH2O and resuspend RNA pellet
Store at minus 30°C overnight before proceeding to the next step
NEXT DAY
Quantify the RNA, determine volume required for 10µg (one gel lane)
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