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Total RNA extraction from yeast cells Emmanuela Marchi PhD Dept.

. Pharmacology UFIR Comparative nutritional systems biology Focus Team Post-doc emanuela.marchi@unifi.it 16 April 2009 When citing this SOP you should acknowledge both NuGO and the appropriate NuGO partner institution that has made the SOP available. Please use a form of words such as: We used the NuGO Standard Operating Procedure (SOP) number 60 produced by the University ofFlorence. Details of the SOP are available via the web link: http://www.nugo.org/frames.asp?actionID=38869&action=loginFromPP

Reagents and Materials AE Buffer 50mM Sodium Acetate pH 5.5 1mM EDTA To final volume with RNase free water Acid Phenol:Chloroform 5:1 solution pH 4.50.2- Ambion #AM9722 SDS 10% Solution Ambion #AM9822 Chloroform 99.8% A.C.S. SIGMA #366919 Ethanol 96extra pure MERCK #1.00971 Ethanol 70% is prepared as dilution of Ethanol 96% with nuclease free water. Instruments Water bath at 65C. Thermo-block at 65C. Microcentrifuge at 4C. Fume hood. NanoDrop 1000 Spectrophotometer - Thermo Scientific (Sodium Acetate 3M pH 5.5 Ambion #AM9740) (EDTA 0.5M pH 8.0 Ambion #AM9261) (Nuclease Free Water Ambion #4387936)

Pre-procedure Pre-heat a water bath at 65C and warm Phenol-Chloroform. Pre-heat a thermo-block at 65C. Cool a centrifuge at 4C.

Aliquot 50l of 10% SDS in a number of microcentrifuge tubes corresponding to the number of samples.

Clean all the working area and materials with RNaseZap (Ambion - # AM9782).

Main Procedure Always work on ice with gloves! 1. Harvest yeast cells by centrifuging at 4C (samples can be frozen in liquid nitrogen and stored at -80C). 2. Working on ice, resuspend the pellet in 450l of AE buffer. Transfer each sample in an appropriately labelled microcentrifuge tube containing 50l 10% SDS and briefly vortex. 3. Add 500l of pre-heated Acid Phenol:Chloroform and vortex for 10 seconds. 4. Incubate at 65C for exactly 10 minutes and vortex each sample every minute. During this incubation keep Acid Phenol:Chloroform in the heated water bath. 5. Incubate at least 5 minutes on ice. 6. Centrifuge at 12000-14000 rpm for 5 minutes at 4C. 7. Transfer the upper aqueous phase into a fresh appropriately labelled tube and add an equal volume (500l) of pre-heated Acid Phenol:Choloroform for further purification. 8. Vortex and centrifuge at 12000-14000 rpm for 5 minutes at 4C. 9. Transfer the upper aqueous phase into a fresh appropriately labelled tube and add an equal volume (500l) of Chloroform. 10. Vortex and centrifuge at 12000-14000 rpm for 5 minutes at 4C. 11. Transfer the upper aqueous phase into a fresh appropriately labelled tube and add 1/10 volume (50l) of Sodium Acetate 3M pH 5.5 and 2 volume (1ml) of 96% Ethanol. 12. Precipitate over-night at -20C. The day after (Always work on ice with gloves) 13. Centrifuge at 12000-14000 rpm 4C for at least 20 minutes. A with pellet should appear at the bottom of the tube. 14. Remove the supernatant with vacuum or by pipetting and wash pellet with 500l of 70% Ethanol. Only add Ethanol: dont mix! 15. Centrifuge at 12000-14000 rpm at 4C for 10 minutes. 16. Remove the supernatant and let the pellet completely dry on the bench keeping the tubes in ice. Smell the tube in order to understand when Ethanol has evaporate. 17. Add to the dried pellet a volume of RNase free water depending on the pellet dimension (20100l). Let the samples on ice in order to allow water to dissolve the pellet. 18. Briefly vortex and centrifuge spin. 19. Store at -80C.

Sub Procedures: RNA quantification Prepare a 1:10 dilution of each sample (i.e. 2l sample + 18l water) and use 1-2l for NanoDrop quantification. We consider as good sample those having OD260/OD280 ratio near 1.8-2.0. RNA integrity control Prepare an 1% agarose gel added with Ethidium Bromide 500ng/ml. Load about 0.5-1g RNA and reach a final volume of 10l adding formamide. Let the gel run at 50-80V for 30 minutes. Good RNA are characterized by two well defined bands corresponding to the rRNAs 18s and 28s.

Safety When using Phenol and Chloroform work under fume hood. Always wear gloves for protecting yourself from reagents and the samples from RNases present on your hands. Always keep sample on ice in order to prevent nucleases activation.

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