Magnesium chloride (MgCl
MW 95.23)
| 1 mM | Dissolve 95.2 mg of magnesium chloride per final volume of 1 liter. |
| 4 mM | Dissolve 0.381 grams of magnesium chloride per final volume of 1 liter. |
| 10 mM | Dissolve 0.952 grams of magnesium chloride per final volume of 1 liter. |
| 0.1 M | Dissolve 9.523 grams of magnesium chloride per final volume of 1 liter. |
| Note: | A single stock solution of 1 M MgCl can be mixed by dissolving 95.23 grams of magnesium chloride to a final concentration of 1 liter with water, and all dilutions made appropriately from this stock solution. |
Magnesium sulfate (MgSO
MW 120.39)
| 5% (w/v) | Dissolve 5.0 grams of magnesium sulfate to a final volume of 100 ml with water or buffer. |
Mayer's hematoxylin
Purchase commercially or mix with either of the following procedures:
A. Dissolve 1.0 gram of hematoxylin in 10 ml of absolute ethanol. Dissolve 20 grams of potassium alum (KAl(SO) · 12HO) in 200 ml of water. In a chemical hood, with protection against explosion, bring the potassium alum solution to a boil and add hematoxylin/ethanol mixture. Continue to boil for approximately 1 minute. Add 0. 5 grams of mercuric oxide and cool rapidly. Add 0.5 ml of glacial acetic acid. Filter before use. This mixture is stable for about two months.
B. Alternatively: Dissolve 5.0 grams of hematoxylin in 50 ml of absolute ethanol and add to 650 ml of warm water. Heat gently until the hematoxylin dissolves and then add 300 ml of glycerin, 0.3 grams of sodium iodate and 20 ml. of glacial acetic acid. Co ol and make volume up to 1 liter with distilled water. Filter before use.
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>-Mercaptoethanol (MW 78.13)
| 0.5 M | Density = 1.2 grams/ml. Use either 3.91 grams OR 3.26 ml of mercaptoethanol in a final volume of 100 ml of water or buffer. |
| 5% (w/v) | Use 5.0 grams or 4.167 ml in a final volume of 100 ml of water or buffer. |
MES (2-(N-Morphilino)ethanesulfonic acid MW 195.2)
| 0.1 M | Dissolve 1.952 grams of MES to a final volume of 100 ml with water or buffer. |
Methanol (CH
OH MW 32.04)
| 22 M | The methanol can be weighed (704.9 grams) or measured volumetrically by using the density. That is, 704.9 grams  0.7914 grams/ml or 890.7 ml of methyl alcohol. Weigh or measure the appropriate amount and dilute to a final volume of 1 liter with water. |
Methanol/Acetic Acid (for fixing proteins in acrylamide gels)
| 45%:12% | Add 120 ml of glacial acetic acid to 450 ml of methanol and dilute to a final volume of 1 liter with water. |
Methanol/Acetic Acid (for destaining or fixing proteins in acrylamide gels)
| 5%:7% | Add 70 ml of glacial acetic acid to 50 ml of methanol and dilute to a final volume of 1 liter with water. |
Methyl green
| 0.2% (w/v) | Dissolve 0.2 grams of methyl green to a final volume of 100 ml with 0.1 M acetate buffer, pH 4.2. Acetate buffer (0.1 M pH 4.2) is prepared by dissolving .361 grams of sodium acetate (trihydrate) in approximately 80 ml of water. Add .42 ml of glacial acetic acid and adjust the volume to 100 ml with water. |
Microtubule buffer (MT buffer)
Dissolve 19.52 grams of MES in 800 ml of distilled water. Add 0.380 grams of EGTA and 47.62 grams of MgCl. Adjust the pH to 6.4 with HCl or NaOH and dilute to a final volume of 1 liter with disti lled water.
Minimum essential medium (MEM)
For all purposes of this manual, MEM refers to Eagle's MEM. While it is possible to mix this medium, it is infinitely easier (and less expensive) to purchase the media pre-mixed from any number of commercial sources (Gibco, Flow, KC Biological, Sigma Chem ical). The ingredients are listed in Table 12.1. It is essential that chemicals of the highest purity are used throughout.
NG agar (Nematode Growth agar)
Dissolve 3.0 grams of NaCl, 2.5 grams of peptone, and 17 grams of agar in a final volume of 1 liter. Boil to dissolve the agar, autoclave to sterilize.
Meanwhile, prepare separate sterile solutions of:
Using proper sterile technique, cool the agar solution slightly and add 1 ml of CaCl, 1 ml of uracil, 0.5 ml of cholesterol, 25 ml of phosphate buffer, and 1 ml of MgSO. Swirl to mix all ingredients and pour plates.
-Nitrophenyl phosphate (MW 263.1)
| 0.05 M | Dissolve 1.32 grams of -nitrophenyl phosphate to a final volume of 100 ml with water or buffer. |
| 0.8% (w/v) | Dissolve 0.8 grams of -nitrophenyl phosphate to a final volume of 100 ml of water or buffer. |
| Note: | Sigma Chemical Co., St. Louis, supplies this compound as Sigma 104, Phosphatase Substrate, Cat. # 104-0. |
Osmium tetroxide (OSO MW 254.2)
| 1% | Osmium tetroxide is a gas which is used in solution for EM preservation. It is best purchased in sealed vials of 2 ml of 4% OsO/4/. For use, add 6.0 ml of water or buffer to the 2.0 ml of 4% osmium tetroxide. Seal in a tightly sealed container, wrapped with aluminium foil and keep in the refrigerator. Use of a fume hood is mandatory when using OsO/4/. Osmium tetroxide will rapidly fix the nasal passages and exposed cornea if not properly vented. It should be handled with extreme care. |
Perchloric acid (PCA MW 100.47)
| 2% (w/v) | Dissolve 2.0 grams of PCA to a final volume of 100 ml with water or buffer. |
Percoll
Colloidal PVP coated silica. Available from Sigma Chemical Co., St. Louis. Cat. #P 1644.
Periodic Acid (Periodate Used for PAS reaction)
Dissolve 0.6 grams of periodic acid in 100 ml of water and add 0.3 ml of concentrated nitric acid.
Phenazine methosulfate (PMS MW 306.34)
Mutagen and irritant.
| 0.033% (v/v) | Add 33 μ l of phenazine methosulfate to 90 ml of water or buffer and make up to 100 ml final volume. Must be made immediately prior to use. |
Phenol mixture
Combine 555 ml of aqueous phenol (or 500 grams of phenol crystals plus 55 ml of water) with 70 ml of mcresol. Add 0.5 grams of 8-hydroxyquinoline.
Phenol will cause severe burns and readily dissolves all plastic and rubber compounds. Use extreme caution when handling this compound.
-Phenylenediamene oxalate (PPDO MW 198.18)
| 0.02% (w/v) | Dissolve 20 mg of PPDO to a final volume of 100 ml with water or buffer. |
Phosphate buffered saline (PBS)
Mix 100 ml Ca
/Mg fr ee 10X PBSA with 800 ml of distilled water. Separately, dissolve 0.1 gram of magnesium chloride and 0.1 gram of anhydrous calcium chloride to a final volume of 100 ml with water. With constant stirring, slowly add the magnesium/calcium chloride solution t o the diluted PBSA. If a precipitate forms, start over, and add slower with continuous stirring.
Ca/Mg free Phos phate buffered saline - 10X (10X PBSA)
Dissolve 80 grams of NaCl, 2.0 grams of KCl, 15.0 grams of Dibasic sodium phosphate and 2.0 grams of Monobasic potassium phosphate in 1 liter of distilled water. This makes a 10X solution of Ca/Mg free phosphate buffered saline. Dilute 1:10 prior to use. Store in a refrige rator.
Phosphate buffered saline-Tween 20 (4.8)
Mix PBS and add 0.1% (v/v) Tween 20.