The following is a list of the solutions and chemicals required throughout the laboratory manual. It is organized alphabetically, and individual exercises list the materials needed for that exercise. For many solutions, directions are given for a molar solution and the user is left to dilute to the appropriat e concentration for their needs. There are many vendors of the chemicals listed, and many of the solutions can also be purchased pre-mixed.

Acetic acid (MW 60.05) Glacial acetic acid is 99.6% (w/v) acetic acid, and is 17.4 M.

1 M	Add 57.5 ml of glacial acetic acid to 800 ml of water and then make to 1 liter with water.
0.05 N	Add 2.87 ml of glacial acetic acid to 800 ml of water and then make to 1 liter with water.
0.9 M	Add 54 ml of glacial acetic acid to 800 ml of water and then make to 1 liter with water.
7% (w/v)	Add 70 ml of glacial acetic acid to 800 ml of water and then make to 1 liter with water.
45% (w/v)	Add 450 ml of glacial acetic acid to 500 ml of water and then make to 1 liter with water.

Acetic Acid/Butanol/Water (15:60:25)

Combine 150 ml of glacial acetic acid, 600 ml of nbutanol and 250 ml of water.

Aceto-orcein

Add 2.0 grams of orcein to 45 ml of glacial acetic acid. Bring to a boil and continue to heat until completely dissolved. Cool and add 55 ml of distilled water. Filter prior to use. Note: Some early investigators added a of an iron salt (such as ferric citrate) as a mordant. It tends to increase the intensity of the aceto-orcein stain. The same reaction can be had by chopping plant material with an older steel (not stainless) razor blade.

Acid alcohol

Add 1.0 ml of concentrated HCl to 100 ml of 70% (v/v) ethyl alcohol.

Acid Orcinol Reagent (0.1% FeCl in 10% HCl)

Add 0.1 grams of FeCl to 50 ml of 10% (v/v) HCl and make to 100 ml with 10% HCl.

Acrylamide Solutions

Acrylamide solutions for PAGE are given as total concentration of acrylamide (acrylamide + bisacrylamide) and the amount of cross linker (bisacrylamide). This is listed as the T:C ratio. For example, a 10% gel (10%T:5%C) would contain a total of 10 g rams of acrylamide per 100 ml, and would be composed of 5 grams of acrylamide and 5 grams of bisacrylamide. Usually, a stock solution of 30% acrylamide is produced containing 0.8% bis-acrylamide. Many investigators use 30 grams of acrylamide plus 0.8 gram s of bis-acrylamide per 100 ml of water, but 29.2 grams of acrylamide plus 0.8 grams of bis would be technically correct. In practice, it makes little difference since the gels are diluted to 10% or less. The 30% stock solution is filtered through a 0.45 µ filter and stored at 4 ° C in the dark. For use, the stock solution is diluted with an appropriate buffer (usually a 2X Tris-HCl). The stock solution is stable for about one month. Discard after this period.

Acrylamides in their monomeric form are neurotoxic. Polymerize all acrylamide solutions prior to disposal.

SDS, -mercaptoethanol and a tracker dye (bromophenol blue) are added at various points. Refer to Chapter 4 for more complete details.

Alcian blue (MW 1300)

0.001 M

Dissolve 0.13 grams of Alcian Blue 8GX (Sigma # A-2899) in 100 ml of water.

Alcohol Orcinol Reagent (10% orcinol in 95% ethanol)

Dissolve 1.0 gram of orcinol in 95% ethanol to a final volume of 10 ml .

Alkaline Distilled Water

Add one pellet of NaOH to 1 liter of distilled water.

Alkaline Solution for G-banding

Dissolve 2.8 grams of NaOH and 6.2 grams of NaCl to a final volume of 1 liter with water.

-Aminosalicic Acid (PAS MW 175.1)

6% (w/v)

Dissolve 6.0 grams of PAS to a final volume of 100 ml with water or buffer.

Ammonium acetate (MW 77.08)

0.1 M

Add 7.708 grams of Ammonium acetate to a final volume of 1 liter of water.

Ammonium persulfate (MW 228.2)

10% (w/v)

Dissolve 1.0 grams of ammonium persulfate to a final volume of 10 ml with water. Mix fresh, prior to use as a catalyst for PAGE. Normally, about 50 µ l of ammonium persulfate is added to each 15 of gel solution for polymerization.Dissolve 0.13 grams o f Alcian Blue 8GX (Sigma # A-2899) in 100 ml of water.

Ammonium sulfate (MW 132.14)

2% (w/v)	Add 2 grams of ammonium sulfate to a final volume of 100 ml water.
4.1M(sat.) 0.001 M	Dissolve 542 grams of ammonium sulfate to a final volume of 1 liter.

n-Amyl alcohol (Pentanol C H OH MW 88.15) Density = 0.8144 grams/ml

0.38 M

The amyl alcohol can be weighed (33.5 grams) or measured volumetrically by using the density. That is, 33.5 grams 0.8144 grams/ml or 41.1 ml of n-amyl alcohol. Weigh or measure the appropriate amount and dilute to a final volume of 1 liter with water.

Amylase, buffered pH 7.0

1% (w/v)

Dissolve 0.5 grams of amylase to a final volume of 50 ml with 0.01 M sodium phosphate buffer, pH 7.0.

Ascorbic acid (MW 176.12)

2 mM	Dissolve 35.2 mg of ascorbic acid to a final volume of 100 ml with water.

ATP (Adenosine triphosphate, MW 507.21)

5 mM	Dissolve 254 mg of ATP to a final volume of 100 ml with water or buffer.Dissolve 35.2 mg of ascorbic acid to a final volume of 100 ml with water.

Baker's Formalin Add 1.0 gram of calcium chloride, 1.0 gram of cadmium chloride and 10 ml of concentrated formalin to 75 ml of water. Make to a final volume of 100 ml with water.

Benzoic acid (MW 122.12)

8 mM	Dissolve 98 mg of benzoic acid to a final volume of 100 ml with water or buffer.

Bis-acrylamide (N,N'-Methylene-bis-acrylamide) Cross linker for acrylamide gels. Refer to Acrylamide solutions or Chapter Four for more details.

Biuret Reagent Add 1.50 grams of CuSO ・ H O and 6.0 grams of sodium potassium tartrate to 500 ml of water. Separately make 300 ml of 10% (w/v) NaO H by dissolving 300 grams of NaOH to a final volume of 300 ml with water. Combine the two solutions in a 1 liter volumetric, swirl to mix and make up to 1 liter with water. Store the final solution in a dark, plastic bottle. Discard if black or red precip itate forms.

Bovine Serum Albumin (BSA) There are many grades of BSA available and care should be taken when using this protein. For routine protein concentration standards, a 96-99% pure fraction (Sigma # A 2153) may be used. For tissue culture, RIA, or molecular weight standardization, BSA sh ould be optained which is extracted and purified specifically for that purpose.

1% (w/v)

Dissolve 0.5 grams of BSA to a final volume of 50 ml in water or buffer.

Bradford Protein Assay This procedure uses an absorbance shift in an acidic Coomasie Blue solution. It is commercially available from Pierce Chemical Company, Rockford, Illinois as Protein Assay Reagent, Cat. # 23200. It contains methanol and solubilizing agents and is very rel iable. If you wish to make your own, dissolve 100 mg of Coomasie Brilliant Blue G-250 in 50 ml of 95% ethanol. Add 100 ml of 85% phosphoric acid, and bring to a final volume of 1 liter with distilled water.

Phosphoric acid is extremely corrosive. Handle with care.

Bromophenol blue (Sodium salt, MW 692.0)

0.001 (w/v)

Dissolve 1 mg of Bromophenol blue, sodium salt (Sigma # B7021) to a final volume of 100 ml with either water or buffer.

n-Butanol (C H OH MW 74.12) Density = 0.8098 grams/ml

1.1 M

The butanol can be weighed (81.5 grams) or measured volumetrically by using the density. That is, 81.5 grams 0.8098 grams/ml or 100.7 ml of n-butanol. Weigh or measure the appropriate amount and dilute to a final volume of 1 liter with water.

C. elegans Ringers This is a basic saline solution for nematodes. Dissolve 11.36 grams of Na HPO ・ 7H O, 3.0 grams of KH PO , 0.5 grams of NaCl and 1.0 gram of NH Cl to a final volume of 1 liter. Adjust the pH to 7.0. May be autoclaved for sterilization.

Calcium chloride (MW 110.99)

0.0033 M

Dissolve 0.522 grams of calcium acetate to a final volume of 1 liter with water or buffer.

Calcium chloride (MW 110.99)

0.001 M	Dissolve 0.111 grams of anhydrous calcium chloride to a final volume of 1 liter with water or buffer.
0.08 M	Dissolve 8.879 grams of anhydrous calcium chloride to a final volume of 1 liter with water or buffer.
2% (w/v)	Dissolve 2 grams of anhydrous calcium chloride to a final volume of 100 ml with water or buffer.

cAMP (Adenosine monophosphate, cyclic MW 329.22)

0.001 M (1mM)

Dissolve 33 mg of cAMP to a final volume of 100 ml with water, buffer or media.

Carnoy fixative Combine 10.0 ml of glacial acetic acid with 60.0 ml of absolute ethyl alcohol and 30.0 ml of chloroform.

Chloroplast homogenization buffer To 400 ml of distilled water, add 30.058 grams of sorbitol, 2.23 grams of sodium pyrophosphate, 0.407 grams of magnesium chloride, and 0.176 grams of ascorbic acid. Adjust the pH to 6.5 with HCl and dilute to a final volume of 500 ml.

Chloroplast suspension buffer To 400 ml of distilled water, add 30.058 grams of sorbitol, 0.372 grams of EDTA, 0.102 grams of magnesium chloride and 5.958 grams of HEPES buffer. Adjust the pH to 7.6 with NaOH and dilute to a final volume of 500 ml.

Chrom alum gelatin (Subbing solution) Dissolve 5.0 gram of gelatin in 1 liter of boiling water. Cool and add 0.5 grams of potassium chrome alum (CrK(SO ) ・ 12H O). Store in refrigerator. To use, dip clean slides into the solution and dry in vertical position in a dust free location.

Citric acid (H C H O ・ H O MW 210.14)

0.1 M

Dissolve 21.01 grams of citric acid to a final volume of 1 liter.

Citrate buffer (Sodium phosphate-Citrate buffer)

0.001 M
PH 4.8	Add 493 ml of 0.2 M Na HPO to 507 ml of 0.1 M citric acid.
PH 3.6	Add 322 ml of 0.2 M Na HPO to 678 ml of 0.1 M citric acid.
PH 4.2	Add 414 ml of 0.2 M Na HPO to 586 ml of 0.1 M citric acid.
PH 5.4	Add 557.5 ml of 0.2 M Na HPO to 442.6 ml of 0.1 M citric acid.
PH 6.0	Add 631.5 ml of 0.2 M Na HPO to 368.5 ml of 0.1 M citric acid.
PH 6.6	Add 727.5 ml of 0.2 M Na HPO to 272.5 ml of 0.1 M citric acid.
PH 7.2	Add 869.5 ml of 0.2 M Na HPO to 130.5 ml of 0.1 M citric acid.
PH 7.8	Add 957.5 ml of 0.2 M Na HPO to 42.5 ml of 0.1 M citric acid.

Cobaltous Nitrate (MW 182.96)

2% (w/v)

Dissolve 2.0 grams of cobaltous nitrate (hexahydrate is very soluble) to a final volume of 100 ml with water. Keep well closed in a cool place.

Colcemid

10 µ g/ml

Dissolve 10 g of colcemid per ml of saline or culture medium.

Coomasie blue (Coomasie Brilliant Blue R250)

0.25% (w/v)0.001 M

Dissolve 2.50 grams of Coomasie Brilliant Blue R250 to a final volume of 1 liter with 20% (w/v) trichloroacetic acid (TCA). Some investigators use a 0.25% solution of Coomasie Blue in methanol-water-glacial acetic acid (5-5-1).

Copper sulfate (CuSO 5H O MW 249.68)

0.5% (w/v)

Dissolve 0.13 grams of Alcian Blue 8GX (Sigma # A-2899) in 100 ml of water.

0.5% (w/v) Dissolve 0.5 grams of copper sulfate to a final volume of 100 ml with water.

Copper tartrate/carbonate (CTC) Dissolve 0.5 grams of copper sulfate and 1.0 gram of potassium sodium tartrate to a final volume of 100 ml with water. Combine 1.0 ml of this solution with 50 ml of 2% Na CO in 0.1 N NaOH. Must be made fresh, prior to use. Stock solutions are stable.

Crystal Violet Dissolve 0.1 grams of crystal violet and 0.25 ml of glacial acetic acid to a final volume of 100 ml with water.

DCMU (3-(3,4-Dichlorophenyl)-1,1-Dimethylurea MW 233.1)

1 x 10 M 0.001 M	Dissolve 2.3 mg DCMU to a final volume of 100 ml with water or buffer.
5 x 10 M0.001 M	Dilute the 1 x 10- M solution 1/200 prior to use.

Dichlorophenolindophenol (DCPIP MW 290.1)

0.0025 M	Dissolve 73 mg of DCPIP to a final volume of 100 ml with water or buffer.
0.0001 M	Dissolve 2.9 mg of DCPIP to a final volume of 100 ml with water or buffer.

Dinitrophenol (DNP MW 184.11)

18.4 mg%

Dissolve 18.4 mg of 2,4-dinitrophenol to a final volume of 100 ml with water or buffer.

Dische diphenylamine reagent Dissolve 500 mg of diphenylamine in 49 ml of glacial acetic acid. Add 1.0 ml of concentrated HCl.

Dithiothreitol (Cleland's Reagent MW 154.3)

0.01 M

Dissolve 154 mg of dithiothreitol to a final volume of 100 ml with water or buffer. Dithiothreitol is available from Sigma Chemical Co., St. Louis, Cat # D0632. Dithioerythritol may be substituted.

DOPA (3-(3,4-Dihydroxyphenyl)-L-alanine MW 197.19)

8 mM	Dissolve 158 mg of L-DOPA to a final volume of 100 ml with water or buffer. Note that the maximum solubility of DOPA in water is 165 mg/100 ml (8.3 mM).

EDTA (Ethylenediaminetetraacetic acid MW 292.24)

1 M

Dissolve 292.24 grams of EDTA, free acid to a final volume of 1 liter. If the more soluble disodium salt of EDTA is used, adjust the weight accordingly. The pH can be adjusted with acetic acid or NaOH. For corresponding concentration dilutions, multiply the weight in grams by the desired molarity. For exam ple, for 10 mM EDTA, multiply 292.24 X 0.010 to obtain 2.92 grams of EDTA per liter.

EGTA (Ethylene Glycol-bis( aminoethyl Ether) N,N,N',N'-Tetraacetic Acid MW 380.4)

1 mM	issolve 380 mg of EGTA to a final volume of 1 liter with water or buffer.

Eosin

0.5% (w/v)

Dissolve 0.5 grams of Eosin Y in 100 ml of water.

Ethanol (C H OH MW 46.07) Density = 0.7893 gm/ml

50-95% (v/v)