>> 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.
>> I'm glad protocol works well for you, Carolyn. Here is a
> reposting of the method.
>>> 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
>> 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
> 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
> 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.
> Verwoerd, TC, Dekker, BMM, Hoekema, A (1989). A small-scale
> procedure for the rapid isolation of plant RNAs. NAR 17: 2362