Useful buffers |
Biological buffers
Tris buffer
The established protocol for ensilication requires 50 mM of Tris buffer at pH 7.0 with no sodium present. Therefore, 3.03 g of Trizma-Base (MW: 121.14 g/mol, cat: T666-1kg, Sigma UK) was weighed out to make 500 ml buffer. In a beaker, 400 ml of ultrapure (MilliQ, MilliPore UK) water was added to the weighed Trizma and using a magnetic stirrer this was dissolved. The pH was set using 32% (v/v) hydrochloric acid. Finally, the solution was filled up to 500 ml final volume with ultrapure water.
Recombinant expression in E. coli
Bacterial medium, Luria Broth (LB) contains 10 g/L Tryptone, 5 g/L Yeast Extract, 5 g/L Sodium Chloride, Sigma, in ddH2O. Autoclave after preparation and use when needed.
Agar plates were made of LB, 10 g/L Tryptone, 5 g/L Yeast Extract, 10 g/L Sodium Chloride, with 15 g/L added agarose, Sigma, in ddH2O. Autoclave after preparation and pour plates when hand warm in a laminar flow-hood.
Bacterial stocks were made by addition of 30% glycerol and stored at -80 °C until further use.
Buffers for HisTag purification:
NaCl | Tris | Imidazole | |||
IMAC25 (binding) | 0.5 M | 0.1 M | 25 mM | pH 8.0 in ddH2O. | |
IMAC500 (elution) | 0.5 M | 0.1 M | 500 mM | pH 8.0 in ddH2O. |
pH solution with HCl. Autoclave or filter 0.45 μm for use in the FPLC.
ELISA 5’-5’ tetra-methyl-benzidine (TMB, Sigma, UK) substrate
TMB | NaAct | H2O2 | ||
stock | 10 μg / ml | 0.1 M pH 6.0 | 30 % (v/v) | TMB in DMSO or EtOH NaAct stock 1M pH with citric acid. |
in 10 ml | 100 µL | 10 ml | 1.5 µL |
5’-5’ tetra-methyl-benzidine (TMB, Sigma, UK) was dissolved in 1 ml of dimethylsulfoxide (DMSO, Sigma, UK) or EtOH at 10 μg / ml and 100 μL was added to 10 ml of 0.1M sodium acetate pH 6.0 (with citric acid) with addition of 1.5 μL of 30% (v/v) H2O2.
Sodium Phosphate Buffers
pH | Volume (mL) of 1 M Na2HPO4 | Volume (mL) of 1 M NaH2PO4 |
---|---|---|
5.8 | 7.9 | 92.1 |
6.0 | 12.0 | 88.0 |
6.2 | 17.8 | 82.2 |
6.4 | 25.5 | 74.5 |
6.6 | 35.2 | 64.8 |
6.8 | 46.3 | 53.7 |
7.0 | 57.7 | 42.3 |
7.2 | 68.4 | 31.6 |
7.4 | 77.4 | 22.6 |
7.6 | 84.5 | 15.5 |
7.8 | 89.6 | 10.4 |
8.0 | 93.2 | 6.8 |