Wendel Lab
Department of Ecology, Evolution and Organismal Biology
Iowa State University, Ames, IA 50011, USA

Prior to Extractions

• Bake all necessary glassware, metal spatulas, mortars, and pestles overnight in a 200°C oven after wrapping them in aluminum foil. For each sample, you should minimally prepare one mortar and pestle, one homogenizer and one spatula. DEPC-treat a sufficient volume of water for all dilutions (50 mL is usually plenty). 
• Prepare all reagents and solutions. 

Day 1

1. Place all mortars to be used in liquid nitrogen to cool. Heat homogenizers to 65°C and acquire dry ice. After the mortars are cooled, nestle them in the dry ice. Collect up to 0.1 g of tissue and flash freeze in liquid nitrogen. Transfer to a cooled mortar and pestle. Grind tissue until it is a fine powder in liquid nitrogen. Remove one pre-warmed glass homogenizer. Add 800µL of the 85°C XT buffer and 1 µL of Proteinase K solution (10 mg/mL). Transfer the frozen, ground sample to the warm XT buffer and grind thoroughly in glass homogenizer until the suspension is evenly dispersed. Transfer homogenate to a labeled eppendorf tube. Mix and incubate for 1.5 hours in the 42°C incubator/shaker. Remove from incubation. Add 104 µL of 2M potassium chloride (for a final concentration of 160 mM; this step will precipitate proteins from the extract). Gently vortex to mix samples. Incubate on ice for 1 hour. Centrifuge at max rpm for 20 minutes at 4°C to remove debris. Transfer the supernatants to new, labeled eppendorf tubes. 
2. Add 1/3 volume of 8M lithium chloride (making a final concentration of 2M). Incubate on ice overnight (~333µL). 

Day 2

1. Place tubes in the pre-chilled centrifuge and chilled rotor. Spin at max rpm for 20 minutes at 4°C. Decant and discard supernatant. Wash pellet in 500µL of ice-cold 2M lithium chloride. Disperse pellet with a pipette tip (as needed) after adding the lithium chloride. This will minimize the retention of unwanted substances. Centrifuge tubes at max rpm for 15 minutes at 4°C. Decant and discard supernatant. Repeat steps 2 & 3 twice more. Spin for 6 minutes. Suspend pellet in 25 µL of 1X TE, ph 8.0 and gently vortex. The sample may be warmed to room temperature to facilitate diffusion. Remove the insoluble material by centrifuging tubes at max rpm for 10 minutes at 4°C. Save the supernatant and transfer it to pre-labeled 1.5 mL microcentrifuge Eppendorf tubes. Add 1/10 volume (about 3µl) of 2 M potassium acetate (pH 5.5). Incubate on ice for 15 minutes. This will removed positively-charged polysaccharides, residual proteins, and other salt-insoluble material. Centrifuge tubes at max rpm for 15 minutes at 4°C. Discard pellet by transferring supernatant to a new tube. Precipitate RNA with 1/10 volume (about 3.5µL) of 3 M sodium acetate (pH 6.0) and 2.5X volume (about 87.5 µL) of cold 100% ethanol. Store at -80°C for 1-2 hours. Centrifuge at max rpm for 20 minutes at 4°C to collect RNA. Remove ethanol and discard. Wash with 100µL of 70% ethanol. Centrifuge for 5 minutes at 4°C. Aspire or pipette off the ethanol. 
2. Resuspend RNA in 10 µL of DEPC-treated water and store at -80°C. Alternatively: To assess the quality of the DNA, prepare a 1% agarose gel (0.30 g agarose in 30 mL 1X TAE buffer). Load 1 µL of each RNA sample into each well after mixing the aliquot with fast orange tracking dye. Run a ladder in one lane. The gel should be electrophoresed at 40-50 volts, stained in ethidium bromide, and visualized under an ultraviolet light source. For even clearer gels, use the formaldehyde electrophoresis protocol. 

This protocol is a scaled down version of the full-scale micro protocol usually used. For additional information see that protocol. 

Reagents and Solutions

DEPC-Treated Water
< !> Perform all these steps under a fume hood!

Add 0.05% (v/v) DEPC to the required volume of distilled, deionized water (for example, 500 mL of water requires the addition of 250µL DEPC). Mix by vigorous stirring for approximately 30 minutes. Autoclave to break down the DEPC. 

Hot Borate Extraction Buffer (XT Buffer)

* = add just before use. To dissolve, add Borax, EGTA, SDS, and sodium deoxycholic acid to pre-warmed deionized, distilled water. Adjust pH to 9.0 with sodium hydroxide. DEPC-treat and autoclave.

For 38ml aliquot of XT Buffer:
DTT 0.6g
PVP 0.73g
IGEPAL 360µl

• Proteinase K (10 mg/mL in DEPC-treated dd-water; store stock at -20°C)
• 1X TE (10 mM Tris-Hcl, pH 7.5 with 1 mM EDTA, pH 8.0)
• 2 M Potassium chloride
• 2 M Potassium acetate, pH 5.5
• 2 mM AND 8 mM Lithium chloride (keep in refrigerator)
• 3 M Sodium acetate, pH 6.0
• Ethanol, 70% and 100% (keep at -20°C)
• Liquid nitrogen (for tissue grinding)

Protocol updated May 2003. Many thanks to Ryan Rapp.