Homogenization step

1. Weigh the tissue
2. Add Trizol: 1ml per 50-100mg tissue used
3. Homogenize
4. Incubate samples for 5 min at room temp
5. Add chloroform: 2000µl per 1ml of trizol used initially
6. Mix thoroughly
7. Incubate 2-3 min at room temp
8. Centrifuge 15min at 11400 rpm (11338rpm=12000G) and 2-8°C

Aqueous phase contains the RNA

(Note: All aqueous phase must be removed for retrieval of high quality DNA)

1. Remove aqueous phase for RNA extraction (see RNA extraction protocol)
2. Precipitate DNA from with ethanol: 300µl 100% ethanol per 1ml of trizol used initially
3. Mix samples carefully and incubate 2-3min at room temp
4. Centrifuge 5min at 4600rpm (4629rpm=2000G) and 2-8°C

Aqueous phase contains the protein

• Remove aqueous phase for protein extraction
• Precipitate proteins with isopropanol (2-propanol): 1.5ml per 1ml trizol used initially
• Incubate 10min at room temp
• Centrifuge 10 min at 11400rpm and 2-8°C
• Remove supernate
• Wash pellet 3 times in 0.3M Guanidine Hydrochloride in 95% ethanol (wash solution

1.                 Add 2ml wash solution per 1ml Trizol used initially
2.                 Incubate 20min at room temp
3.                 Centrifuge 5min at 9000rpm (8963rpm=7500G) and 2-8°C

• Remove supernate
• Vortex protein pellet in 2ml ethanol
• Incubate 20 min at room temp
• Centrifuge 5min at 9000rpm (8963rpm=7500G) and 2-8°C
• Vaccum dry pellet 5-10min
• Dissolve pellet in 1% SDS by pipetting and possibly incubate at 50°C
• Centrifuge 10min at 10350 (10350=10000G) and 2-8°C to sediment insoluble material
• Transfer supernate to fresh tube
• Ready to use/ quantify by ?

Erik Edström & Mikael Altun, 010614; updated 020625