Biochemistry Reagents and Stock Solutions

Buffer Solutions

PBS (1 L)

 

Bacterial Cloning and Expression Stocks

Ampicillin (50 mg/mL)

  1. Place 1 g ampicillin in 20 mL dH2O.
  2. Sterilize by passing through a 0.2 µm filter.
  3. Aliquot and freeze at -20 °C.

Dilute your 50 mg/mL stock 1:500 into growth media to obtain the usual 100 µg/mL antibiotic restriction.

 

IPTG (1M)

  1. Dissolve 2.83 g IPTG in 8 mL dH2O. Bring to 10 mL with additional dH2O.
  2. Sterilize by passing through a 0.2 µm filter.
  3. Freeze at -20 °C.

Induction of protein expression is usiually accomplished with an IPTG concentration of 0.5-1 mM.

 

Lysozyme (25 mg/mL)

  1. Place 0.5 g lysozyme in 20 mL dH2O.
  2. Aliquot and freeze at -20 °C.

To break down the cell wall for lysing bacteria, a final concentration of 0.25 mg/mL lysozyme (1:100 dilution) is added to the pelleted and resuspended cells in lysis buffer.

 

DNaseI (2 mg/mL)

  1. Place 20 mg DNase I in 10 mL 20% glycerol, 75 mM NaCl.
  2. Aliquot and freeze at -20 °C.

To digest DNA during bacterial lysis, add to a final concentration of 10 µg/mL in lysis buffer, with 5 mM MgCl2.

 

Resuspension Buffer (for inclusion body purification)

For resuspension of pelleted bacterial cells, use 10 mL resuspension buffer per L of culture.

 

Lysis Buffer (for inclusion body purification)

After resuspension of bacteria, add 2.5 X the volume of resuspended cells of lysis buffer, along with lysozyme, DNase, and MgCl2. Let stir at rm. T. 30 min.

 

Common Strengths of Commercial Reagents

 Reagent MW Molar Concentration  mL/L for 1 M Solution
Acetic Acid, Glacial

60.05

17.4

57.5

Hydrocloric Acid

36.6

11.6

86.2

Nitric Acid

63.02

15.99

62.5

Sulfuric Acid

98.1

18.0

55.6

Ammonium Hydroxide

35.0

14.8

67.6

Sodium Hydroxide

40.0

19.1

52.4

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