PBS (1 L)
Bacterial Cloning and Expression Stocks
Ampicillin (50 mg/mL)
Dilute your 50 mg/mL stock 1:500 into growth media to obtain the usual 100 µg/mL antibiotic restriction.
Induction of protein expression is usiually accomplished with an IPTG concentration of 0.5-1 mM.
Lysozyme (25 mg/mL)
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)
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||
|Welch 4.264 The University of Texas at Austin Austin, TX 78712 (512) 471-3279|
Inquiries? Suggestions? Contact: firstname.lastname@example.org