Reagent Preparation

Note: All reagents are made with double distilled H2O unless otherwise noted.

Stock Solutions

In molecular biology, we commonly work in relatively small volumes (less than a ml). This creates a problem for making solutions from scratch every time we use them. For example, the working concentration for MgCl2 in Hind III digest is 10 mM. The molecular weight of MgCl2 is 203.30, so to make 10 µl of 10 mM MgCl2 you would need

203.3 g/×L0.01M×0.000 01 L = 2.3×105 g

of MgCl2.

To get around the problem of weighing out such a small amount, we usually make buffers and many other solutions at concentrations 10 times (10X) or greater than needed. We then simply add the appropriate volume to the reaction we are setting up, to get the desired final concentration. For example, restriction enzyme buffers are usually made as a 10X stock. The working concentration is usually 1X (but always check the instructions for a particular enzyme – occasionally it will be used at a concentration other than 1X).
This means you need to regularly calculate dilution factors. See below for some practice problems.

1 M Tris

Dissolve 121.1 g of Tris-HCl in 800 ml of H20. Adjust the pH to the desired value by adding concentrated HCl.

desired pH amount of conc HCl
7.4 70 ml
7.6 60 ml
8.0 42 ml

Check pH using a Tris compatible electrode, and adjust. Bring volume to 1 L. Dispense in 100 ml aliquots and autoclave for 15 min at 15 psi on liquid cycle.

0.5 M EDTA

Add 186.1 g of disodium EDTA•2H20 to 800 ml of H20. Add 20 g of NaOH pellets.
Stir until all the NaOH has dissolved (EDTA will not go into solution until pH is close to 8.0). Adjust the pH to 8.0.
Dispense into 100 ml aliquots and autoclave for 15 min at 15 psi on liquid cycle.

5 M NaCl

Dissolve 292 g of NaCl in 800 ml of H20.
Adjust volume to 1 liter with H20.
Dispense into 100 ml aliquots and autoclave for 15 min at 15 psi on liquid cycle.

10 M NaOH

Add 40 g of NaOH slowly to 80 ml of H20. Stir until completely dissolved.
Bring volume to 100 ml and store in a plastic bottle. There is no need to autoclave.

NOTE: This is an exothermic reaction; the beaker will become quite hot and can break. Use plastic beakers as a precaution.

SDS (10% w/v)

Dissolve 100 g of SDS in 900 ml of H20. Heat gently and stir to aid dissolution.
Dispense into 100 ml aliquots and store at RT. Do not autoclave.

1 M CaCl2•2H20 - for competent cell production

Dissolve 14.7 g of CaCl2•2H20 in 80 ml of H20. Bring to volume, filter sterilize.
Dispense in 10 ml aliquots and store at –20 ºC.
For transformation prepare 0.1 M CaCl2 fresh by thawing a 10 ml aliquot of 1 M CaCl2, adding 90 ml of dd H20 and filter sterilizing.

Media and antibiotics

LB-broth (Luria-Bertani Broth)

Place a magnetic stir bar in a 1 L flask. Weigh and add the following:

  • 10 g NaCl
  • 10 g tryptone
  • 5 g yeast
  • 800 ml of H2O

Stir until components have dissolved.
Adjust the pH to 7.0 with 5 N NaOH
Adjust the volume to 1 L.
Transfer to appropriate containers for sterilizing by autoclaving.

SOB media

Per liter:

  • 20 g tryptone
  • 5 g yeast extract
  • 0.5 g NaCl
  • 800 ml H2O

Add 10 ml of 250 mM KCl (made by dissolving 1.86 g of KCl in 100 ml H2O). Adjust pH to 7.0. Bring the volume to 1 L. Sterilize by autoclaving.
Just before using add 5 ml of sterile 2 M MgCl2 per L (made by dissolving 19 g of MgCl2 in a final volume of 100 ml H2O, and sterilized by autoclaving).

SOC media (SOB media with 20 mM glucose)

After autoclaving SOB media, cool to 60 ºC or less. Add MgCl2 and 20 ml of sterile 1 M glucose (18 g of glucose in a total volume of 100 ml, filter sterilized). Aliquot and freeze at -20 ºC until needed.

Agar plates

To make agar plates from any media

  • Follow the procedure for making the liquid media.
  • After dispensing into desired containers add agar at the rate of 15 g/L.
  • Sterilize by autoclaving (agar does not dissolve until heated).
  • Add antibiotics as required.
  • Pour approximately 20 ml per plate.

Antibiotic containing media

name [stock] [working]
Ampicillin 50 mg/ml in H2O 20 – 50 μg/ml
Kanamycin 10 mg/ml in H2O 10 – 50 μg/ml
Tetracycline 5 mg/ml in ethanol 10 – 50 μg/ml

Sterilize by filtering through a 0.22 μM filter, except if dissolved in ethanol.
Before adding antibiotics to media, let the media cool to 50 ºC.

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