Clarification of "RNA isolation Protocols"

DELANEYT 919-541-8577 DELANEYT at am.abru.cg.com
Thu Jun 10 02:45:11 EST 1993


To the readers of ARABIDOPSIS at NET.BIO.NET,  

   I would like to clarify that the RNA isolation protocol resent out from GUT at qucdn 
incorrectly stated that the procedure was developed "By Terry Delaney."  That language was 
inserted by GUT at qucdn; my original message began with the statement:

      "Here is a protocol we use with good success:"
   
   I don't take credit for the protocol, but did provide it in response to GUT at qucdn's 
request (the protocol is adapted from: Verwoerd et al, NAR 17: 2362).  In addition, my 
response to was also edited by GUT at qucdn to include the word "approximately" in several 
places, which was not in my original posting.  
   I do appreciate the effort taken by users of the bulletin board in sharing the replies to 
their queries with the network, but encourage a minimum of editing to preserve the intent and 
content of the original reply.  I have attached my original response to GUT at qucdn's request 
below:

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In Response to: RNA ISOLATION PROTOCOL WANTED (posted by GUT at qucdn on 6/2/93):
From: delaneyt at am.abru.cg.com (reply later on 6/2/93):
==============================================================================

Here is a protocol we use with good success:

RAPID Arabidopsis RNA PREPS

1. Take leaf sample (one leaf sufficient if plant is quarter-sized or larger) at 3-4 weeks 
after planting, place into liquid nitrogen filled eppindorf and grind using drill (and 
plastic pestle (e.g. Kontes)), keep on dry ice until ready to extract, or store at -200C.

2. Add 500 ul 800C 1:1 phenol (water saturated) to extraction buffer, vortex.
     EXTRACTION BUFFER: 100 mM LiCl, 100 mM tris pH 8.0, 10 mM EDTA, 1.0 % SDS.

3. Add 250 ul chloroform, vortex.

4. Spin five minutes or more.

5. Take aqueous phase and add to 1/10 volume of 3.0 M NaOAc, then add 2 volumes ETOH, place 
on ice or at -200C, approx 30 minutes.

6. Spin 10 minutes in microfuge, dry pellet

7. Resuspend in water (for one leaf use approx 5.5 ul, plus 19.5 loading mix, and load half 
onto gel (unless very small plant, then use all).

        
LOADING MIX (per sample): 
12.5 ul formamide/BFB/XC*,
4.25 ul formaldehyde,    
2.5 ul MSE buffer,      
0.1 ul EtBr (10 mg/ml).             
   *formamide/BFB/XC: 10 mg bromophenol blue, 10 mg xylene cyanol, 100 g formamide
        
RNA GEL: 260 ml water, 30 ml 10X MSE, 3.6 g agarose, 9.0 ml formaldehyde (add in hood after 
melted  gel has cooled to touch).
                        
10X MSE:                        
200 mM MOPS,    
50 mM NaOAc,    
10 mM EDTA, 
    pH to 7.0 with NaOH, autoclave, (turns yellow).

Good luck,

Terry Delaney 
Department of Molecular Genetics
Ciba Geigy Agricultural Biotechnology
Research Triangle Park, NC 
919-541-8577
e-mail: delaneyt at am.abru.cg.com

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