Various Chemicals

The Phage Cookbook by Dominik Refardt

Various Chemicals

Arabinose

The molecular weight of L-arabinose is 150.13, and for a 1 M solution you need therefore approximately 30 g in 200 mL H2O. Which incidently gives you also a 15% solution (weight/volume). Sterilize by filtering or by autoclaving.

Mitomycin C (Fluka 69824-1EA)

For prophage induction. Dissolve content of the bottle (2 mg) in 2 mL 70% ethanol (to obtain a concentration of 1 μg/μL). This stuff is supposedly very nasty, so be careful when dissolving and aliquoting it. Use a syringe with a long needle to penetrate the lid of the bottle and add the ethanol. Gently swirl the bottle to dissolve the mitomycin C and then use the syringe again to empty the bottle and distribute the mitomycin to 1.5-mL tubes. Make 500 μL aliquots. Store at -20 °C. Stays fine for at least a year that way.

When filling ethanol into the bottle through the rubber lid, you will build pressure inside the bottle and if you don't release it (by pulling the plunger back), the syringe may spray mitomycin C all over the place when you remove it from the bottle. So be careful.

SDS (sodium lauryl (or dodecyl) sulfate)

10% or 20% (weight per volume) SDS are usually used. To prepare a 10% solution, dissolve 5 g SDS in 50 ml water.

    • If you want to do it properly: 200 g of electrophoresis-grade SDS in 900 ml of H2O.

    • Heat to 68 °C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl.

    • Adjust the volume to 1 L with H2O. Store at room temperature.

    • Sterilization is not necessary. Do not autoclave.

Use a mask when weighing this out or work in the fume hood! Don't shake it to dissolve.

Sodium acetate

Used in DNA precipitation protocols. Stock concentration is 3 M.

    • Dissolve 24.6 g sodium acetate, anhydrous, in 100 mL H2O.

    • Adjust pH to 5.2 with glacial acetic acid.

X-Gal

Stock: 40 mg/ml in DMSO or DMF. Wrap in aluminium foil and store at -20 °C. DMF dissolves some plastic materials and is toxic. DMSO needs to be thawed when stored in the freezer.

Working concentration: 40-80 µg/mL.

Spread 40 µL directly on the plate or add it to the liquid agar before pouring.

IPTG

Stock concentration: 0.1 M (molecular weight of IPTG is 238 g/mol). Working concentration: 0.1 mM, i.e. 100 µM. Sometimes, to get really strong induction, you might want to double this to 200 µM.

Dissolve 0.24 g in 10 mL double-distilled water, filter sterilize, store at -20 °C.

Add to the liquid agar before pouring.

Alternatively, you can spread it directly on a plate. 25 µL on a plate containing 25 mL agar (which is a thick layer) give ample induction.