RNA prep (Verwoerd et al)

[DELANEY 919-541-8577]

A few daze ago, Carolyn M. Wetzel wrote:

>On 15 Sept 1994, Terry Delaney posted a protocol for 
>rapid RNA preps from Arabidopsis.  I've used it quite 
>successfully.  Perhaps you could post it again, Terry, if you if 
>you still have it on file?  
>Otherwise, for the person who asked:  see Verwoerd et al., 
(1989) NAR 17:  2362.
>
>-Carolyn
>

I'm glad protocol works well for you, Carolyn.  Here is a 
reposting of the method.  

-Terry
-- 
       

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 -20°C.

2. Add 500 ul 80 C 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 -20 C, approx 30 
minutes.

6. Spin 10 minutes in microfuge, dry pellet

7A. Resuspend in water (for one leaf use approx 5.5 ul; 

7B. Or to reprecipitate, and clean up further, do a LiCl 
precipitation:
        Resuspend in water:     320 ul
        Add 8M LiCl             200 ul  (3M LiCl final)
        ON @ 5 C or -20 C for a few hours.
        Pellet, wash with 80% ETOH.
        Resus in water (5.5 ul or more for larger sample)
Will later add 19.5 loading mix, and after heating (65°C 10') 
load half onto gel (unless very small plant, then use all).


LOADING MIX             
(per sample):                     or for 20 samples:                             
12.5 ul formamide/BfB/XC*               250 ul                          
4.25 ul formaldehyde                     85 ul                          
2.5 ul   10x MSE buffer                  50 ul                           
0.1 ul   EtBr (10 mg/ml)                  2 ul                                              

*formamide/BfB/XC:                                                      
10 mg bromophenol blue                                                  
10 mg xylene cyanol                                                     
100 g formamide

RNA GEL (300 mls):      
260 ml water                            
30 ml 10X MSE buffer                            
3.6 g agarose                           
9.0 ml formaldehyde (add in hood after melted                                                   
gel has cooled to touch)
                                                                
For 1000 mls 10X MSE buffer:               
200 mM MOPS     41.86 g free acid
50 mM NaOAc     16.6 ml 3.0 M (pH = 5.2)
10 mM EDTA      20 ml 0.5 M
                                        
pH to 7.0 with NaOH, autoclave, turns yellow.

Reference:
Verwoerd, TC, Dekker, BMM, Hoekema, A (1989). A small-scale 
procedure for the rapid isolation of plant RNAs.  NAR 17: 2362