Rasayan - Preparation of common chemicals

2007-05-19T17:42:00.000+05:30

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.

2007-05-19T17:38:00.000+05:30

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.

2007-05-19T17:35:00.000+05:30

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.

2007-05-19T17:31:00.000+05:30

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.

2007-05-19T17:26:00.000+05:30

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.

2007-05-19T17:24:00.000+05:30

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.

2007-05-19T17:13:00.000+05:30

Proteinase K buffer (10X)

100mM tris-Cl (pH 8.0)
50mM KCl
30mM MgCl2

2007-05-19T17:10:00.000+05:30

NaOH (10N)

The preparation of 10N NaOH involves a highly exothermic reaction, which can cause breakage of glass contain. Prepare this solution with extreme care in plastic beakers.

To 800ml of distilled water, slowely add 400g of NaOH pellets, stirring continuously.

As an added precaution, place the beaker on ice. When the pellets have dissolved completely adjust the volume to 1 litre with water. Store the solution in a plastic container at room temperature.

Sterilization is not necessary.

2007-05-05T16:28:00.000+05:30

Lysozym (10mg/ml) stock

Dissolve solid lysozyme at a concentration of 10mg/ml in 10mM tris-Cl (pH 8.0) immediately before use. Make sure that the pH of the tris-Cl solution is 8.0 before dissolving the protein.

Lysozyme will not work efficiently if the pH of the solution is less than 8.0.

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2007-05-05T16:26:00.000+05:30

Gel loading buffer (60X stock)

0.3% (W/v) Bromophenol blue
0.3% (W/v) Xylene Cyanol FF
30% (V/v) Glycerol
600mM EDTA
100mM Tris-HCl (pH 7.6 – 8.0)

Store the stock solution in -200C for long-term preservation.

To prepare 6X solution use 30% glycerol to dilute the stock. This 6X stock solution can be stored at 40C.

In 1% agarose gels, bromophenol blue migrates with 300bp fragment whereas xylene cyanol FF migrates with the 4000bp fragment.
The volume ratio of sample to loading dye solution should be 5:1, respectively.

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2007-05-05T16:23:00.000+05:30

Ethidium Bromide solution (10mg/ml)

Add 1g of Ethidium bromide to 100ml of distilled water stir on a magnetic stirrer for several hrs wrap the container in Aluminium foil.

Store at room temperature.

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2007-05-05T16:18:00.000+05:30

TBE Buffer

For stock solution (10X) for One liter:
Tris Base : 108g
Boric acid : 55g
Na2.EDTA.2H2O : 7.44g

Adjust the pH 8.0

Working solution is 0.5X and which contains 45mM Tris-Borate and 1mM EDTA

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2007-05-05T16:14:00.000+05:30

EDTA (0.5M, pH 8.0)

Add 186.1gm of disodium EDTA.2H2O to 800ml of distilled water. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20g of NaOH pellets).

Dispense into aliquots and sterilize by autoclaving.

The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

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2007-05-05T15:58:00.000+05:30

Calcium Chloride

Dissolve 11g of CaCl2.6H2O in a final volume of 20ml of distilled water.

Sterilize the solution by passing it through a 0.22µM filter.

Store in 1ml aliquots at 4 0C.

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2007-05-05T15:41:00.000+05:30

Ammonium Acetate (10 M)

To prepare a 1 liter solution, dissolve 770g of ammonium acetate in 800ml of water. Adjust the volume to 1 liter with water. Sterilize by filtration.

Sterilize the solution by passing it through a 0.22-micrometer filter. Store the solution in tightly sealed bottles at 4 oC or at room temperature.

Ammonium acetate decomposes in hot water and solutions containing it should not be autoclaved.

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