Department of Biological Sciences,

1000 Hilltop Circle

Baltimore, MD 21250

Prepare a 5x stock solution in 1 liter of H2O:

54 g of Tris base

27.5 g of boric acid

20 ml of 0.5 M EDTA (pH 8.0)

The pH of the concentrated stock buffer should be approx. 8.3.. Some investigators prefer to use more concentrated stock solutions of TBE (10x as opposed to 5x). However, 5x stock solution is more stable because the solutes do not precipitate during storage. Passing the 5x or 10x buffer stocks through a 0.22μm filter can prevent or delay formation of precipitates.

TE buffer:

10 mM Tris-Cl (pH, usually 7.6 or 8.0)

1 mM EDTA (pH 8.0)

Use concentrated stock solutions to prepare. If sterile water and sterile stocks are used, there is no need to autoclave. Otherwise, sterilize solutions by autoclaving for 20 minutes. Store the buffer at room temperature.

1 M Tris-Cl – used at various pHs

Using Tris base : To make 1 liter, dissolve 121 g Tris Base in 800 ml of water. Adjust pH to the desired value by adding approximately the following:

 

pH = 7.6 about 60 ml of concentrated HCl

pH = 8.0 about 42 ml of concentrated HCl

Make sure solution is at room temperature before making final pH adjustments. Bring final volume to 1 liter. Sterilize by autoclaving.

Using Trizma tables: an alternate procedure for preparing Tris solutions is to combine the proper amount of Tris Base and Tris Hydrochloride to achieve the desired value using Sigma's Tris tables.

WB (10% redistilled glycerol, 90% distilled water, v/v)

In a 1-liter graduated cylinder, add 100 ml of glycerol and 900 ml of distilled water. Cover with parafilm and mix thoroughly. Sterilized by autoclaving, and chill to 4°C.

Western Blotting Solutions:

1X Transfer buffer 1: 25 mM Tris, 192 mM Glycine, pH 8.3

Mix 3.03 g Tris, 14.4 g glycine; add dd water to 1 liter – do not adjust pH.

1X Transfer buffer 2: 25 mM Tris, 192 mM Glycine, pH 8.3, 20 % methanol

Mix 3.03 g Tris, 14.4 g glycine; add 200 ml methanol; add dd water to 1 liter – do not adjust pH. (NOTE: methanol is not needed for PVDF membranes)

10X Western Buffer: 200 mM Tris pH = 7.5; 1.5 M NaCl

To prepare 1X Western Buffer, dilute 10X buffer to 1X, adding Tween-20 to 0.1%. Remove 50 ml and set aside for the last two washes. To the remainder, add I-Block to 0.2%, heating gently with constant stirring until dissolved. Bring to room temperature before using.

X-gal 5-bromo-4-chloro-3-indolyl-b-D-galactoside (same recipe for X-phosphate)

Make a 2% (w/v) stock solution by dissolving X-gal in dimethylformamide at a concentration of 20 mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to prevent damage by light and store at -20 ° C. It is not necessary to sterilize X-gal solutions.

This Web page is maintained by Julie B. Wolf, UMBC;
Last updated on 3/2/2010