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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 Recipes Supplies Tips (责任编辑：泉水) |
Isogen RNA Extraction Protocol
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
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