RNA isolation Protocols

GUT at QUCDN.QUEENSU.CA GUT at QUCDN.QUEENSU.CA
Sat Jun 5 15:36:00 EST 1993


Dear netters:

Since we posted the message regarding RNA isolation from
Arabidopsis, too many requests for the protocols have been
received to send them on a individual basis. Therefore, we are
posting them on the network for your reference. we must apologize
that some of the protocols that we have received were
inadvertently lost from the VM mainframe. For your convenience
and reference, we will list the names and e-mail addresses of
those who have supplied the following protocols. Please note that
the comments in the protocols are made by the authors. If you
have any questions, please contact them directly. Again, we wish
to thank all those whose supplied us protocols and apologize for
not being able to list all of them. Tie-Sheng Ali.

RNA isolation from plant tissue
     Modified from Biotechniques 8:148 by Cancade Timpte and P.
     Green, E-mail: ctimpte at bio.indiana.edu
(For rosette leaves, I can get a mg of RNA per gram of leaf. For
etiolated seedlings, 100 ug per gram)
1. Grind 0.5-1 gr tissue under liquid nitrogen with swirling
motion, with 3 additions of N2.

2. Disperse powdered tissue into 30 ml oak ridge tube  containing
5 ml GTC buffer, vortex, hold on ice.

3. Add 0.5 ml 2 M NaOac pH 4.0, vortex.

4. Add 5 ml phenol, shake or vortex 2 min.

5. Add 1 ml CHCl3, shake or vortex 2 min.

6. Centrifuge 10,000 xg 10 min at 4C.

7. Remove aqueous phase (Should have big interphase, do not take
any of it. Transfer to corex tube. You may wish to back extract
with more phenol, it may increase yield slightly.)

8. Add 5 ml isopropanol, precipitate on ice 10 min.

9. Centrifuge 10,000 xg 10 min 4C.

10. Carefully pour off supernatant(pellet may be dime size and
may slide on wall. Invert tubes and drain to dry.)

11. Transfer pellet to eppendorf by resuspending in 1 ml 4 M
LiCl.

12. Vortex 5 min. (If pellet is big, repeat with 0.5 ml LiCl.)

13. Resuspend in 0.5 ml SDS-TE, vortex 5 min (You may need to
triturate with pipettor to disperse pellet.)

14. Phenol:Chloroform extract. vortex 5 min, spin 5 min. (If
pellets are small, just extract with chloroform.)

15. Chloroform extraction (Avoid interface!).

16. Add 1/10 volume 3 M NaOac pH 5.0, 600 ul isopropanol,
precipitate on ice 10 min.

17. Sin 10 min, dry in speed vac. Resuspend in 100-500 ul of
water. (For preps from hypocotyl, use 100 ul.)

Buffers (I do not DEPC treat any of my buffers or water,
autoclaving is sufficient.)

GTC-----200 ml
4 M guanidinium isothiocyanate
25 mM Na Citrate pH 7.0---5 ml 1 M stock
0.5% sarkosyl------20 ml 10% stock (N-laroyl sarkosine)
0.1 M mercaptoethanol---1.4 ml
mix guanidine, water and citrate, add sarkosyl after guanidine is
solubilized.
DO NOT autoclave.

5 X MOPS----liter
0.1 M MOPS pH 7.0---20.93 g
40 mM NaOAc----3.28 g
5 mM EDTA-----10 ml 0.5 M stock
(I do not autoclave this buffer either.)

1% Agrose gel
1.5 g agrose
90 ml water
boil, cool to 60 to 70!C
add 30 ml 5 X MOPS, 27 ml 37% formaldehyde
Pour gel and ENJOY!

Sample preparation
Dry samples in speed vac, resuspend in 2 ul water, 2 ul 5X MOPS,
10 ul formamide, 3.5 ul formaldehyde, 1 ul 0.1 mg/ml ethidium
bromide. Boil 1 min, quick chill on ice before loading onto gel.
(I run my gels 4 hr at 60 volts in the recirculating buffer box
and get adequate separation. Blot in 10 X SSC without any prior
treatment.)
RAPID Arabidopsis RNA PREPS

By Terry Delaney
Department of Molecular Genetics
Ciba Geigy Agricultural Biotechnology
Research Triangle Park, NC
e-mail: delaneyt at am.abru.cg.com

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 eppendorf and grind using drill (and plastic
pestle(e.g., Kontes)), keep on dry ice until ready to extract. or
store at -20C.

2. Add 500 ul 80C 1;1 phenol (water-saturated) to extraction
buffer, vortex. (Extraction buffer: 100 mM LiCl, 100 mM Tris pH
8.0, 10 mM EDTA, approximately 1.0% SDS).

3. Add 250 ul chloroform, vortex.

4. Spin 5 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 -20C (Minus 20 Celcis.)
for approximately 30 minutes.

6. Spin 10 minutes in microfuge, dry pellet.

7. Resuspend in water (for one leaf use approx 5.5 ul, plus 19.5
loading mix, and load half onto gel (if the plant is very small,
use all in gel electrophoresis).

Loading Mix: 12.5 ul formamide/BFB/XC*, 4.25 ul formaldehyde,
about 2.5 ul MSE buffer, -0.1 ul EtBr (10 mg/ml)
*Formamide/BFB/XC: 10 mg bromophenol blue, 10 mg xylene cyanol,
100 g formamide.
RNA gel: 260 ml water, about 30 ml 10 X MSE, about 3.6 g agrose,
about 9 ml formaldehyde (Add in fume hood after agrose is melted
and cooled down (touchable with hand)).

10 X MSE: 200 mM MOPs, about 50 mM NaOAc, about 10 mM EDTA. pH to
7.0 with NaOH, autoclave (the solution turns yellow).

RNA preparation from Arabidopsis leaves

Lynne Reuber
MGH (Dr. Ausubel lab)
Phone (617)726-5959
Fax   (617)726-5949
E-mail: Reuber at frodo.MGH.Harvard.EDU

1. Grind 0.1-0.2 g frozen leaves in liquid nitrogen in a mortor
with s pestle.

2. Scoop powder into an eppendorf tube containing 0.4 ml RNA
extraction buffer and 0.4 ml water-saturated phenol, vortex well
and put on ice.

3. Repeat 1 and 2 until the eighteenth sample. Vortex each tube
once when a new sample is added onto ice.

4. Spin for 5 min, transfer 0.5 ml supernatant to eppendorf tubes
containing 0.5 ml chloroform, vortex well.

5. Spin for 5 min, transfer 0.4 ml supernatant to eppendorf tubes
containing 1 ml 100% EtOH and 40 ul 3 M DEPC-treated NaOAC, mix
well and put on dry-ice for 10 min.

6. Spin for 10 min, remove EtOH carefully.

7. Add 0.4 ml 2M DEPC-treated LiCl. Try to dissolve pellets by
pipetting up and down.

8. Spin 10 min, remove supernatant. Resuspend in 100 ul DEPC-
treated water. Add 10 ul 3 M DEPC-treated NaOAC and 250 ul 100%
cold (-20C) EtOH, mix well.

9. Spin 10 min, remove ETOH, resuspend in 50 ul DEPC-treated
water.

10. Take 1 ul to 0.5 ml water to measure OD260/OD280, 1 OD260=30
ug/ml.
   



More information about the Arab-gen mailing list