BioProtocols--sol3

1. Weight out 3.09 g DTT and dissolve in 15 ml of distilled water.

2. Make up volume to 20 ml with 0.01 M sodium acetate (pH 5.2).

3. Sterilize by filtration through a 0.22 micron filter.
<CRITICAL STEP> Do not autoclave DTT or solutions containing DTT.
<STORAGE> Store in 1 ml aliquots at -20 C.

0.5 M EDTA (pH 8.0)(1 liter)

1. Weigh out 186.1g Na2EDTA.2H2O.

2. Add 800 ml distilled water and stir vigorously on a magnetic stirrer.

3. Adjust to pH 8.0 with NaOH (~20g of NaOH pellets).
<CRITICAL STEP> The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

4. Make up volume to 1 liter with distilled water.

5. Dispense into appropriate aliquots and sterilize by autoclaving.
<STORAGE> Store at room temperature.

Ethidium bromide (10 mg/ml)(100 ml)

1. Weight out 1g ethidium bromide.
<CAUTION> Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn when working with solutions that contain this dye, and a mask should be worn when weighing it out.

2. Make up volume to 100 ml with distilled water.

3. Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved.
<STORAGE> Wrap the container in aluminium foil or transfer the solution to a dark bottle and store at 4 C.
<CAUTION> After use, these solutions should be decontaminated. Refer to institutional guidelines for details on how to do this.

2 x HEPES-buffered saline (100 ml)

1. Weigh out 1.6 g NaCl, 0.074 g KCl, 0.027 g Na2HPO4.2H2O, 0.2 g dextrose, and 1 g HEPES.

2. Add 90 ml distilled water.

3. Adjust to pH 7.05 with 0.5 M NaOH.

4. Make up volume to 100 ml with distilled water.

5. Sterilize the solution by filtration through a 0.22 micron filter.
<STORAGE> Store in 5 ml aliquots at -20 C.

1 M Isopropylthio-?-D-galactoside (IPTG)(10 ml)

1. Weigh out 2 g IPTG.

2. Add 8 ml distilled water to dissolve.

3. Make up volume to 10 ml with distilled water.

4. Sterilize by filtration through a 0.22 micron disposable filter.
<STORAGE> Store in 1 ml aliquots at -20 C.

1 M Magnesium acetate (MgOAc)(1 liter)

1. Weigh out 214.46 g MgOAc.4H2O.

2. Add 800ml distilled water to dissolve.

3. Make up volume to 1 liter with distilled water.

4. Sterilize by filtration.
<STORAGE> Store at room temperature.

1 M MgCl2 (1 liter)

1. Weigh out 203.3g MgCl2.6H2O.
<CRITICAL> MgCl2 is extremely hygroscopic. Buy small bottles (e.g. 100g) and do not store opened bottles for long periods of time.

2. Add 800 ml distilled water to dissolve.

3. Make up volume to 1 liter with distilled water.

4. Dispense into aliquots and sterilize by autoclaving.
<STORAGE> Store at room temperature.

Phenol:chloroform

1. Mix equal amounts of phenol and chloroform.
Phenol is highly corrosive and can cause severe burns. Wear gloves, protective clothing, andsafety glasses when handling phenol. All manipulations should be carried out in a chemical hood. Any areas of skin that come into contact with phenol should be rinsed with a large volume of water and washed with soap and water. Do not use ethanol.

2. Equilibrate the mixture by extracting several times with 0.1 M Tris (pH 7.6).
<STORAGE> Store the equilibrated mixture under an equal volume of 0.01m Tris (pH 7.6) at 4 C in dark glass bottles.

10 mM Phenylmethylsulfonyl fluoride (PMSF)(10 ml)

1. Weigh out 17.4 mg PMSF.
<CAUTION> PMSF is extremely destructive to the mucous membranes of the respiratory tract, the eyes, and skin. It may be fatal if inhaled, swallowed, or absorbed through the skin. In case of contact, immediately flush eyes or skin with copious amounts of water. Discard contaminated clothing. Aqueous solutions of PMSF can be safely discarded after they have been rendered alkaline (pH>8.6) and stored for several hours at room temperature.
<CRITICAL> PMSF is inactivated in aqueous solutions. The rate of inactivation increases with pH and is faster at 25 C than at 4 ?C. The half-life of a 20 ?M aqueous solution of PMSF is about 35 minutes at pH 8.0
If necessary, stock solutions can be prepared in concentrations as high as 17.4 mg/ml (100 mM).

2. Make up volume to 10 ml with isopropanol.
<STORAGE>Aliquots of appropriate volume should be stored at -20 C.

1 M Potassium acetate (pH 7.5)(100 ml)

1. Weigh out 9.82g potassium acetate.

2. Add 90 ml distilled water to dissolve.

3. Adjust to pH 7.5 with 2 M acetic acid.

4. Make up volume to 100 ml with distilled water.
<STORAGE> Aliquots of appropriate volume should be stored at -20 C.

3 M Sodium acetate (pH 7.0)(1 liter)

1. Weigh out 408.1 g sodium acetate.3H2O.

2. Add 800 ml distilled water to dissolve.

3. Adjust to pH 5.2 with glacial acetic acid.

4. Make up volume to 1 liter with distilled water.

5. Dispense into aliquots and sterilize by autoclaving.
<STORAGE> Store at room temperature.