RNA AND PROTEIN EXTRACTION FROM THE SAME SAMPLE
This protocol was modified from the following referece: Bar-Peled, M., A.S. Conc
| This protocol was modified from the following referece: Bar-Peled, M., A.S. Conceicao, L. Frigerio, and N.V. Raikhel. 1995. Expression and regulation of aERD2, a gene encoding the KDEL receptor homolog in plants and other genes encoding proteins involved in ER-Golgi vesicular trafficking. Plant Cell 7: 667-676. | |
| PREPARE SOLUTIONS | |
| 1. Extraction buffer: | 100mM Tris-HCl pH 8; 1% SDS, 1% deoxycholate-sodium, 20 mM EDTA (2% PVP 40,000-optional when using tissues with high conconcentration of phenols) |
| 2. Phenol: | Add to 200 mL liquified phenol 200 mg of antioxidant 8-hydroxyquinoline (final concentration: 0.1 %) Add 200 mL of 1M Tris-HCl pH 8 stir for 15 min allow the phases to separated and discard the upper aqueous phase. Repeat again (the pH of phenolic phase ~ 8). discard the upper aqueous phase and add 100 mL of 0.1M Tris-HCl pH 8. Store in dark bottle at 4o C for 1-4 month |
| 3. Phenol: Chloroform: Isoamyl alcohol (PCI) (25:24:1) (50 mL): | Mix 25 mL of equilibrated saturated phenol, 24 mL of chloroform and 1 mL of isoamyl alcohol. Store with 0.1M Tris-HCl pH 8 up to 1 month (in dark at 4o C) |
| 4. 100% and 70 % cold ethanol: | Prepare by mixing ethanol with dH2 O. Store at -20o C |
| 5. 3M Sodium acetate pH 5.2 (500 mL): | Dissolve 123 g of anhydrous sodium acetate ,CH3 COONa (Merck), in 300 mL dH2 O. Adjust to pH 5.2 with acetic acid, and bring to final volume of 500 mL with dH2 O (Autoclave) |
| 6. 10M LiCl: | Dissolve 21 g of LiCl in 50 mL of dH2 O. Make fresh every 2 weeks |
| 7. 2M KCl: | Dissolve 14.9 g of KCl in 100 mL of dH2 O |
| 8. 0.1M PMSF: | Dissolve 174.2 mg in 10 mL of methanol |
| PROCEDURE | |
| 1. Grind frozen or fresh tissues (up to 1 g) to a fine powder using a mortar and pestle under liquid nitrogen. Add 4.5 mL of extraction buffer and 10 mL of b -mercaptoethanol. Mix with the pestle and pour into a 15 mL centrifuge tube. Depending on the kind of tissue, extra extraction buffer and b -mercaptoethanol may be required | |
| NOTE: for protein analysis: remove ~0.5-1.0 mL into eppendorf tube containing 1-5 m L of 0.1M PMSF. Mix at 4o C, centrifuge for 10 mins at 1000 xg, transfer suppernatant to new tube, and store at -80o C | |
| 2. Add an equal volume of PCI and vortex for 2-5 mins (tubes can be placed on a shaker at 4o C up to 2 hours while extracting other samples). Centrifuge at 12,000 xg for 20 min at 4o C | |
3. Transfer the upper phase with pipette into a clean 15 mL tube. (normally a white band separate between the aqueous and the organic phase) | |
| NOTE: for DNA analysis by PCR remove 0.1 mL and store at 4o C | |
| 4. The RNA-containing aqueous phase is mixed gently with 0.35 mL of 2M KCl (to a final concentration of 0.16M KCl), and the tube is placed at 4o C for 1 hour (0.0875 mL of 2M KCl per mL of homogenate) | |
| 5. Centrifuge at 12,000 xg for 20 min (4o C). Pour the supernatant into a 15 ml tube | |
| 6. Precipitated RNA with LiCl by adding 1/4 vol. (1.1 mL) of 10 M LiCl, (a white precipitate appears) mix gently. Place tube at -20o C for over night or at -80o C for 30 min. Allow the sample to thaw and centrifuge at 12,000 xg for 20 min at 4o C. Aspirate off the (yellowish color) supernatant using vacuum drawing | |
7. Wash the white RNA pellet from salts with ~5 mL cold 70% EtOH, mix gently (Not by vortex). Carefully aspirate off the supernatant using vacuum drawing (If the pellet dislodges, spin for 3 mins at 12,000 xg at 4o C). The pellet is clear white. Do not dry the pellet by speed vac | |
| 8. Dissolve the pellet with 1 mL of dH2 O, and precipitate by adding 0.1 mL of 3M Na-acetate (pH 5.2) and 2.8 mL of cold ethanol. Mix, and place tubes at 4o C for 15 min (or over night at -20o C) | |
| 9. Centrifuge at 12,000 xg for 20 min at 4o C. Aspirate off the (yellowish color) supernatant using vacuum drawing | |
| 10. Wash the white RNA pellet from salts with 2-5 mL cold 70% EtOH mix gently (Not by vortex). Carefully aspirate off the supernatant using vacuum drawing (If the pellet dislodges spin for 3 mins at 12,000 xg at 4o C). The pellet is clear white. Do not dry the pellet by speed vac | |
11. Dissolve RNA in ~200 mL dH2 O and transfer to 1.5 mL tubes. If RNA is difficult to dissolve: freeze, thaw pellet twice and heat it to 37o C for 2 mins. Store RNA at -80o C |
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