Plant Virus Protocols - Virus extraction for RT-PCR and NASH
RNA extraction for RT-PCR
Adapted from:
Hughes and Galau. 1988. Preparation of RNA from cotton leaves and pollen. Plant Mol. Biol. Rep. 6:253-257.
1. Prepare extraction buffer:
200 mM Tris pH 8.5
1.5% SDS
300 mM LiCl
1% sodium deoxycholate
1% Igepal CA-630
10 mM EDTA
Autoclave the extraction buffer. Just before use add beta-mercaptoethanol (BME) to 0.5%. Use BME in a fume hood.
2. Grind 100 mg of tissue in 500 ul of buffer.
3. Incubate the sample at 65C for 10 minutes.
4. Add 500 ul of 6M potassium acetate pH 6.5
5. Mix sample by inversion, then incubate on ice for 10 minutes.
6. Centrifuge for 10 minutes at high speed.
7. Transfer 650 ul of supernatant to a new microfuge tube and add 1 volume of isopropanol.
8. Incubate for 1 hour at -20C or 30 minutes at -80C.
9. Centrifuge for 10 minutes at high speed.
10. Wash the nucleic acid pellet with 70% ethanol.
11. Resuspend the nucleic acid in 20 ul of sterile water and proceed to RT-PCR.
Membrane viral nucleic acid prep for RT-PCR
Singh, R. P., A. D. Dilworth, M. Singh, and D. L. McLaren. 2004. Evaluation of a simple membrane-based nucleic acid preparation protocol for RT-PCR detection of potato viruses from aphid and plant tissues. J. Virological Methods 121:163-170.
1. Macerate tuber, sprout, or leaf tissue to obtain 150 ul of sap.
2. Add 300 ul SSB plus 0.5% XL-80N triton to the sap. SSB is 10 mM Tris-HCl pH 7.4, 0.65% sodium sulfite.
3. Centrifuge the mixture briefly and spot the supernatant onto nitrocellulose membrane (BA-S 85, pore size 45 um, from Schleicher & Schuell).
4. The viral RNA is stable on the membranes for several weeks. For RT-PCR, elute the RNA from the membrane with 30 ul of water at 80C for 10 min.
TRIZOL Extraction from plant tissue (German Method)
1. Bake ceramic mortars and pestles overnight in oven.
2. Remove mortars and pestles and place on lab bench to cool. Be careful - they are very hot!
3. Cool mortars and pestles with liquid nitrogen. Add plant tissue and more liquid nitrogen and grind plant tissue.
4. Transfer plant tissue to epi-tube with sterile spatula.
5. Add 1 ml Trizol and vortex for one minute.
6. Incubate 5-10 minutes at 40C.
7. Add 200 ul chloroform and vortex for 1 min.
8. Incubate on bench for 1 min.
9. Centrifuge at about 8000 x g for five min.
10. Transfer aqueous phase to new epitube.
11. Add and equal volume of phenol:chloroform (no iso-amyl alcohol).
12. Vortex for 1 min.
13. Centrifuge at about 8000 x g for five min.
(keep the tubes on ice for all of the following steps)
14. Transfer the aqueous phase to a new epitube. Be very careful to avoid the interface where the protein has collected.
15. Add 1/20 vol. of NaCl or 1/10 vol of 3M NaOAc pH 5.0. Prepare these solutions with DEPC water and autoclave before use.
16. Add 2.5 vol. of 100% ethanol and incubate at -20C for at least 1 hour.
17. Incubate tube at room temp for 1 min., then centrifuge at max speed at room temp for 30 min.
18. Wash with 70% ethanol twice. Use fresh, room temperature 70% ethanol, not cold ethanol. Also, be careful since the pellet will be loose in the tube.
19. Air dry the pellet for a few minutes. Do not use a speed vac.
Determine RNA quality in the spec by making a 1:200 dilution and measuring the absorbance at 260 , 270, and 280. The 260/280 ration should be 1.7 or higher. If the absorbance at 270 is higher than 260, then there is phenol in the RNA prep.
RNA concentration in ug/ml = (absorbance at 260)(200)(40ug/ml)