P-MEXP-1952 - P-MEXP-1952

Type
nucleic_acid_extraction
Description
Materials: 1. 3 M Sodium acetate, pH 5 with glacial acetic acid 2. TRIzol-Reagent: 38% Phenol (in saturated buffer) 800 mM Guanidinium thiocyanate 400 mM Ammonium thiocyanate 100 mM Sodium acetate (from 3M stock, pH 5) 5% Glycerol prepare the solution daily in the required quantities; don't use old TRIzol! 3. High salt solution: 1,2 M Sodium chloride 800 mM Sodium citrate 4. Isopropanol 5. Chloroform-Isoamylalcohol 24:1 6. 75% Ethanol (non-denatured) 7. RNase-free water 8. liquid nitrogen 9. Arabidopsis tissue samples (100 – 200 mg in 2 mL tubes) Protocol: 1. grind 100 – 200 mg of Arabidopsis tissue samples under liquid nitrogen to a fine powder and put it back into the 2 mL tube. Don't allow the sample to thaw! 2. add 1000 µL TRIzol 3. vortex until the sample is thawed completely and gives a homogenous mixture. From now on you can work at room tempereature. 4. incubate for 5 min, RT 5. centrifuge at 16.000 ×g, 5 min, 4 °C 6. transfer supernatant into a fresh 2 mL tube 7. add 400 µL Chloroform-Isoamylalcohol 24:1 8. vortex briefly or shake vigorously by hand until the mixture looks homogenous 9. incubate for 5 min, RT 10. centrifuge at 16.000 ×g, 15 min, 4 °C 11. transfer 700 µL of the upper phase to a fresh 1.5 mL tube, without taking anything of the two other phases (rather take less of the upper phase) 12. add 350 µL Isopropanol 13. add 350 µL high salt solution (mixture turns cloudy) 14. mix by repeated inversion until the mixture turns clear again 15. incubate for 10 min, RT 16. centrifuge at 12.000 ×g, 10 min, 4 °C 17. remove supernatant completely by pipeting (use yellow tip if necessary) 18. add 900 µL 75% Ethanol and vortex briefly 19. centrifuge at 7500 ×g, 5 min, 4 °C 20. remove supernatant completely by pipeting (use yellow tip if necessary) 21. add 900 µL 75% Ethanol and vortex briefly 22. centrifuge at 7500 ×g, 5 min, 4 °C 23. remove supernatant completely by pipeting (use yellow tip if necessary) 24. dry pellet at max. 60 ° C until it turns clear. Don't overdry the pellet! 25. dissolve pellet in 30 – 40 µL RNase-free water (incubation for 5 min, 60 ° C helps a lot) 26. take a 1:100 dilution in TE for photometric measurement at 260, 280, and 230 nm 27. calculate the amount of RNA and the ratios 260/280 and 260/230 28. take a sample of 1 µg total RNA for a test gel 29. perform an RNA cleanup with Qiagen RNeasy mini columns according to the manufacturer's instructions 30. the RNA is suitable for amplification by in-vitro transcription if R260/280 > 1,950, and R260/230 > 2,050, and no significant amount of ethidium bromide fluorescence remains in the gel slots, and the RNA is not degraded. Note that the ratios work only with TE buffered dilutions!
Parameters
Amplification, Extracted product
Links
All experiments using protocol P-MEXP-1952: (E-CAGE-5, E-CAGE-8, E-CAGE-9)