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Arabidopsis RNA preps

DELANEYT 919-541-8577 DELANEYT at am.abru.cg.com
Thu Sep 15 08:13:39 EST 1994

To: Richard Dana
c.  arabidopsis at net.bio.net


Here are two protocols that work well for preparing mini and midi 
scale RNA preps from Arabidopsis.

Terry Delaney
Ciba Agric Biotech
Research Triangle Park, NC
delaneyt at am.abru.cg.com

Adapted from:
Verwoerd et al (1989)  NAR 17: 2362

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 
2. Add 500 ul 80 C 1:1 phenol (water saturated) to extraction 
buffer, vortex.
				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, or
7B. 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.

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):			20 samples:
				12.5 ul formamide/BPB/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

RNA sample should be in approx 5 ul (2-10 ug RNA)

							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)

	10X MSE buffer				For 1000 mls (=10X)
    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.

Adapted from Lagrimini et al (1987) PNAS 84: 7542

1. For each sample, label a 30 ml autoclaved Corex tube and add:
        5 ml extraction medium
        5 ml H2O saturated phenol 
    Per sample, put 1 mortar, 1 pestle and 1 spatula 
      (autoclaved) on dry ice. 
    Get dewar with liq N2.

2. Grind 1-2 g frozen tissue finely in mortar.
        Add tissue to mortar, add a little liq N2, 
         let evaporate and grind.
        Important: max. 2g, otherwise yield decreases.
        Grind for a minute after tissue appears fine.

3. Transfer powder with cooled spatula to Corex tube.
        Mix with spatula.

4. Add 5 ml CHCl3. 
        Mix with spatula.

4a. Optional: Polytron sample for 45 sec at setting 5.5

5. Spin in swinging bucket rotor: 10 min 6000 rpm
        Sorvall HB-4 or HS-4.

6. Transfer aqueous phase to new baked Corex tube, 
   record vol. on label and 
   transfer label to new tube. 

7. Add: 1/10th vol. 3M NaAc, pH 5.3 (autoclaved)
                  2 vol. EtOH, -200 C
        Parafilm and mix by inversion.
        Incubate on ice for 30 min.

8. Spin as above: 10 min @ 6000 rpm.

9. Decant sup and drain pellet for 30 min.
        Put tubes in rack at an angle; do not overdry.

10. Touch top of inverted tube to Kimwipe.
       Add 4 ml ddH2O (autoclaved)to pellet.
        Vortex repeatedly and keep on ice.  If neccesary, 
         use 1 ml pipetman to resuspend. OK if small 
         particles are left behind.
        Keep sol'n near bottom when manipulating.

11. Add 2.5 ml 8M LiCl (autoclaved). Parafilm tube, 
        mix and refrigerate o/n.
    Alternatively, place in -80 C freezer for 1 hour, 
        then thaw on ice.

12. Spin as above: 10' @ 6K rpm.

13. Rinse pellet with 1 ml 80% EtOH from -20 C freezer .
    Spin briefly if pellet detaches from tube. 
    Let pellet dry as above.

14. Resuspend pellet in 150 ml ddH2O (autoclaved).
        Keep on ice.
        Hold volume down (or you may have to dry vol. 
        down to get conc. high enough later on)

        If any particulate material is present, spin tube and 
transfer sol'n to new tube.

17. Spec to quantitate

18. Gel electrophoresis as described above.

extraction buffer (Prepare fresh):                                                             
  for 100 ml      
  	     4g   4% p-aminosalicylic acid (Sigma A-3505)                      
        1g   1% 1,5-naphthalenedisulfonic acid, disodium salt  
                 (Kodak 2765)    	
H2O saturated phenol: Add ddH2O to solid phenol. Place in 650C 
waterbath. When fluid, mix well, repeatedly. Store @ room 
temperature. Discard if it turns yellow.

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