May 19, 2007

X-gal solution (2% w/v)

X-gal is 5-bromo-4-chloro-3-indolyl-β-D-galactosidase.

Make a stock solution by dissolving x-gal dimethyl formamide at a concentration of 20mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminium foil to prevent damage by light and store at -200C.

It is not necessary to sterilize x-gal solution by filtration.
Tris-Cl (1M)

Dissolve 121.1g of tris base in 800ml of distilled water. Adjust the pH to the desired volume by adding concentrated HCl.

pH---- HCl
7.4--- 70ml
7.5--- 60ml
8.0--- 42ml

Allow the solution to cool to room temperature before making final adjustments to the pH.

Adjust the volume of the solution to 1 litre with water. Dispense into aliquots and sterilize by autoclaving.

If the 1M solution has a yellow colour, discord it and obtain tris of better quality.

The pH of tris solution is temperature dependant and decreases approx. 0.03 pH units for each 10C increase in temperature.

Tris EDTA (10X)

pH 7.4
100mM Tris-Cl (pH 7.4)
10mM EDTA (pH 8.0)

pH 7.6
100mM Tris-Cl (pH 7.6)
10mM EDTA (pH 8.0)

pH 8.0
100mM Tris-Cl (pH 8.0)
10mM EDTA (pH 8.0)

Sterilize solutions by autoclaving.
Store the solution at room temperature.
Trichloro acetic acid (TCA, 100% Soln.)

To previously unopened bottle containing 500g of TCA, add 227ml of distilled water. The resulting solution will contain 100% (w/v) TCA.
Sodium acetate (3M, pH 5.2 and pH 7.0)

Dissolve 408.3g of sodium acetate.3H2O in 800ml of water. Adjust the pH to 5.2 with glacial acetic acid, or adjust the pH to 7.0 with dilute acetic acid. Adjust the volume to 1 liter with water.

Sterilize by autoclaving.
DNase free RNase
Dissolve pancreatic RNase (RNase A) at a concentration of 10 mg/ ml in 0.01M Sodium acetate (pH 5.2) or in distilled water heat to 1000C for 15 minutes. Allow it to cool slowly room temperature.

Adjust the pH by adding 0.1 volume of Tris-Cl (pH 7.4).

Disperse into aliquots and store at -200C.

RNase precipitates when concentrated solutions are heated to 1000C at neutral pH.
Proteinase K buffer (10X)
100mM tris-Cl (pH 8.0)
50mM KCl
30mM MgCl2