Dr Aswin Doekhie

Useful buffers

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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