Edta Autoclave 20

To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA*2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

Glycerol

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-^m filter. Store in 200-ml aliquots at 4°C.

deionized H2O, to 950 ml tryptone, 10 g yeast extract, 5 g NaCl, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Lysozyme

(10 mg/ml) Dissolve solid lysozyme at a concentration of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately before use. Make sure that the pH of the Tris solution is 8.0 before dissolving the protein. Lysozyme will not work efficiently if the pH of the solution is less than 8.0.

Media Containing Agar or Agarose

Prepare liquid media according to the recipes given. Just before autoclaving, add one of the following:

Bacto Agar (for plates) 15 g/liter Bacto Agar (for top agar) 7 g/liter agarose (for plates) 15 g/liter agarose (for top agarose) 7 g/liter

Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. When the medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose evenly throughout the solution. Be careful! The fluid may be superheated and may boil over when swirled. Allow the medium to cool to 50-60°C before adding thermolabile substances (e.g., antibiotics). To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored markers.

When the medium has hardened completely, invert the plates and store them at 4°C until needed. The plates should be removed from storage 1-2 hours before they are used. If the plates are fresh, they will "sweat" when incubated at 37°C. When this condensation drops on the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread and increases the chances of cross-contamination. This problem can be avoided by wiping off the condensation from the lids of the plates and then incubating the plates for several hours at 37°C in an inverted position before they are used. Alternatively, remove the liquid by shaking the lid with a single, quick motion. To minimize the possibility of contamination, hold the open plate in an inverted position while removing the liquid from the lid.

NaCl

To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at room temperature.

Rich medium

Terrific Broth

For solid medium, please see Media Containing Agar or Agarose. SDS

Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 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 liter with H2O. Store at room temperature. Sterilization is not necessary. Do not autoclave.

10 mM Tris-Cl (pH 8.0) 0.1 M NaCl 1 mM EDTA (pH 8.0)

Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Store the sterile solution at 4°C.

(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Store the buffer at room temperature.

Terrific Broth deionized H2O, to 900 ml tryptone, 12 g yeast extract , 24 g glycerol, 4 ml

For solid medium, please see Media Containing Agar or Agarose.

Shake until the solutes have dissolved and then sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Allow the solution to cool to 60°C or less, and then add 100 ml of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4. (This solution is made by dissolving 2.31 g of KH2PO4 and 12.54 g of K2HPO4 in 90 ml of deionized H2O. After the salts have dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilize by autoclaving for 20 minutes at 15 psi [1.05 kg/cm2] on liquid cycle.)

Tris-Cl

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl.

(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH. Adjust the volume of the solution to 1 liter with H2O. Dispense into aliquots and sterilize by autoclaving.

If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris solutions is temperature-dependent and decreases approx. 0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5°C, 25°C, and 37°C, respectively.

Tris-Sucrose

Sterilize the solution by passing it through a 0.22-pm filter, and store it at room temperature. Solutions containing sucrose should not be autoclaved since the sugar tends to carbonize at high temperatures.

deionized H2O, to 900 ml tryptone, 16 g yeast extract, 10 g

For solid medium, please see Media Containing Agar or Agarose.

To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0 with 5 N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05

kg/cm 2) on liquid cycle.

Was this article helpful?

0 0

Responses

  • vivian donohoe
    Can we add EDTA before Autoclave?
    9 months ago

Post a comment