RNA prep (Verwoerd et al)
DELANEYT at am.abru.cg.com
Tue Nov 29 15:48:04 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.
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
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).
(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
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