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      Powder Mixes for 1 liter of:

LB Broth media formulations have been an industry standard for the cultivation of Escherichia coli since the 1950’s^[1],[2],[3].  These media have been widely used in molecular microbiology applications for the preparation of plasmid DNA and recombinant proteins^[4],[5].  The media are nutrient-rich formulations which provide peptides and peptones, vitamins, and trace elements.  The three formulations differ in the amount of sodium chloride, thus providing selection of the appropriate osmotic conditions for the particular bacterial strain and desired culture conditions.  The low salt formulations, Lennox and Luria, are ideal for cultures requiring salt-sensitive antibiotics such as Zeocin™. 

Formulas – grams per liter

Solid medium contains 15 g bacteriological agar per liter.

Method of Preparation

1.        Dissolve 15.5, 20 and 25 grams, LB-Luria, LB-Lennox, LB-Miller, respectively, in 1 liter of deionized water.  For solid medium, add 15 g bacteriological agar.

2.        Autoclave at 121°C for 15 min.  Store liquid medium at 4°C.

3.        For solid medium, allow to cool to 45°C.  Supplement with antibiotic as needed.  Pour about 30 ml per 100 mm petri dish.  Allow to cure overnight.  Store at 4°C in plastic bags.

 Note:  To promote faster growth, the medium can be supplemented with 10 ml 10% sterile glucose or 40 ml 10% sterile glycerol.

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use and prevent caking of powder.

Turbo Broth™ is used for the cultivation of recombinant strains of E. coli.  This medium has been developed to improve the yield of recombinant proteins.  The carbon source is glycerol which is not metabolized into acetic acid.  It has a rich nutrient base which provides amino acids, vitamins, inorganic and trace minerals at levels higher than those of LB Broth.  The medium is buffered at pH 7.2±0.2 with potassium phosphate to prevent a drop in pH and to provide a source of phosphate.  These modifications permit higher cell yields than can be achieved with LB.  For expression of recombinant proteins, induction should be done at cell densities three times that of LB Broth.  Cell yields are typically 4 to 5 times that of LB Broth in shake flask cultures depending on the characteristics of the strain.  Plasmid copy numbers are higher, which can also enhance protein expression.  This medium is suitable as the base for fermentation cultures.  Biomass yields will depend on the process employed and the strain.  A typical bench-scale fermentation operated in a batch mode can achieve an OD₆₀₀ of 10 to 12 depending on the strain.

Formula – Proprietary

Method of Preparation

1. Dissolve 47.6 grams in 1 liter deionized water.

2. Add 4 ml glycerol.

3. Dispense as required.

4. Autoclave at 121°C for 15 min

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use and prevent caking of powder.

Superior Broth™ is a proprietary medium formulation developed by AthenaES™ for improved expression of recombinant proteins in E. coli.  The medium is a complex, rich formulation which supplies a nitrogen source, vitamins, and moderate glucose levels.  The medium is buffered at pH 7.2±0.2 to prevent acidification of the medium during metabolism of the glucose.  For expression of recombinant proteins, induction should be done at cell densities twice that of LB Broth.  Cell yields are typically about 3 to 4 times that of LB Broth in shake flask cultures depending on the characteristics of the strain.  Plasmid copy numbers are the highest among the AthenaES family of media.  This medium is an excellent choice for large-scale plasmid preparations.  Biomass yields will depend on the process employed and the strain.

Formula – Proprietary

Method of Preparation

1. Dissolve 35 grams in 1 liter deionized water.

2. Dispense as required.

3. Autoclave at 121°C for 15 min.

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use and prevent caking of powder.

Power Broth™ is a proprietary medium formulation developed by AthenaES™ for improved expression of recombinant proteins in E. coli.  The medium is a complex, rich formulation which supplies amino acids, vitamins, and a carbon source at higher levels than Terrific Broth⁵.  The medium is buffered at pH 7.2±0.2.  For expression of recombinant proteins, induction should be done at cell densities three times that of LB Broth.  Cell yields are typically 3 to 4 times that of LB Broth in shake flask cultures depending on the characteristics of the strain.  The amount of soluble protein accumulated for otherwise insoluble proteins is increased by growing in Power Broth™.  This medium is suitable as the base for fermentation cultures.  Biomass yields will depend on the process and the strain employed.

Formula – Proprietary

Method of Preparation

1. Dissolve 52 grams in 1 liter deionized water.

2. Add 4 ml of glycerol.

3. Dispense as required.

4. Autoclave at 121°C for 15 min.

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use, and prevent caking of powder.

Hyper Broth™ is a proprietary medium formulation developed by AthenaES™ for improved expression of recombinant proteins in E. coli.  The medium is a complex, rich formulation which supplies amino acids, vitamins, and glucose at high levels.  This medium produces the highest level of biomass production.  The medium is buffered at pH 7.5±0.2.  This medium has the highest buffering capacity of the expression media set, which permits high glucose levels.  For expression of recombinant proteins, induction should be done at cell densities three times that of LB Broth.  Cell yields are typically 4 to 5 times that of LB Broth in shake flask cultures depending on the characteristics of the strain.  Plasmid copy numbers are higher than for LB, which can facilitate protein expression.  This medium is suitable as the base for fermentation cultures and is excellent for feedbatch applications.  Biomass yields will depend on the process and the strain employed.

Formula – Proprietary

Method of Preparation

1. Dissolve 44.5 grams in 950 ml deionized water.

2. Dispense as required.

3. Autoclave at 121°C for 15 min.

4. Aseptically add 50 ml of Glucose Nutrient Mix.  Mix well

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use, and prevent caking of powder.

Glucose M9Y medium, used to cultivate E. coli, is a minimal medium formulation supplemented with yeast extract.  It is based on M9 salts[6] with a pH of 6.9±0.2 and employs glucose as the carbon source.  Alternative carbon sources may be substituted.  Buffering is provided by a sodium-potassium phosphate system.  Ammonium chloride provides a nitrogen source.  Yeast extract is added to improve the protein production capacity of the medium and to permit cultivation of auxotrophic strains by supplying a source of amino acids, vitamins and minerals.  This differs from the traditional formulation of glucose M9, which employs casamino acids.  Our experience has shown that yeast extract typically gives higher yields of recombinant proteins than casamino acids.

Formula – grams per liter

6.0 g sodium phosphate dibasic                                     4 g glucose

3.0 g potassium phosphate monobasic                           5 g yeast extract

1.0 g ammonium chloride                                              11 mg calcium chloride

0.5 g sodium chloride                                                    241 mg magnesium sulfate

Method of Preparation

1. Dissolve 15.5 grams in 980 ml deionized water.

2. Autoclave at 121°C for 15 min.

3. Allow to cool to below 50°C.

4. Aseptically add 20 ml of Glucose Nutrient Solution.  Mix well.

 ^a Six pack sold as six 500 g containers to ensure freshness during storage, prevent unintentional contamination during use, and prevent caking of powder.

Turbo Prime Broth™ is used for the cultivation of recombinant strains of E. coli.  This medium is made without animal products.  All components are certified "animal product free."  Turbo Prime Broth™ is a derivative of Turbo Broth™ developed to improve the yield of recombinant proteins.  The carbon source is glycerol which is not metabolized into acetic acid, thus improving the pH control of the culture.  It has a rich nutrient base which provides amino acids, vitamins, inorganic and trace minerals at levels higher than those of LB Broth.  The medium is buffered at pH 7.2±0.2 with potassium phosphate to prevent a drop in pH and to provide a source of phosphate.  These modifications permit higher cell yields than can be achieved with LB.  For expression of recombinant proteins, induction should be done at cell densities three times that of LB Broth.  Cell yields are typically 4 to 5 times that of LB Broth in shake flask cultures depending on the characteristics of the strain.  Plasmid copy numbers are higher, which can also enhance protein expression.  This medium is suitable as the base for fermentation cultures.  Biomass yields will depend on the process employed and the strain.  A typical bench-scale fermentation operated in a batch mode can achieve an OD₆₀₀ of 10 to 12 depending on the strain.

Formula – Proprietary