RNA prep (Verwoerd et al)out

Daniel Chamovitz chamo at PEAPLANT.BIOLOGY.YALE.EDU
Tue Nov 29 20:37:14 EST 1994


> 
> 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
> 
> 
> 
> 




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