Back in the Dark Ages when I was in grad school restriction

Back in the Dark Ages when I was in grad school, restriction enzymes didn\'t come with buffers - you had to make up your own. Shown below is the standard stock concentration and the required final concentration of each component in Eco RI storage buffer. How would you prepare 5 ml of this buffer?

Solution

To prepare 5ml of this buffer we require the following quantity of ingredients mentioned:

NaCl 1ml

K2HPO4 75 microlitre

EDTA 1 microlitre

Beta-mercaptoethanol 3.496 microlitre

TritonX-100 12.5 microlitre

BSA 500 microgram

Glycerol 2.5 ml

Explanation:

Using C₁V₁ = C₂V₂

To make a fixed amount of a dilute solution from a stock solution, you can use the formula: C₁V₁ = C₂V₂where:

Incase of NaCl

So, C₁V₁ = C₂V₂

V₁ =(200 *5)/ 1000 mM= 1 ml

Incase of K2HPO4

So, C₁V₁ = C₂V₂

V₁ =(15 *5)/ 1000 mM= 0.075 ml= 75 microlitre

Incase of EDTA

So, C₁V₁ = C₂V₂

V₁ =(0.1 *5)/ 500 mM= 0.001 ml= 1 microlitre

Incase of Beta-mercaptoethanol

So, C₁V₁ = C₂V₂

V₁ =(10 *5)/ 14300 mM= 0.003496 ml= 3.496 microlitre

Incase of TritonX 100

So, C₁V₁ = C₂V₂

V₁ =(0.25 *5)/ 100 = 0.0125 ml= 12.5 microlitre

Incase of BSA

100 microgram/ml

So for 5 ml= 100*5= 500 microgram

Incase of Glycerol

So, C₁V₁ = C₂V₂

V₁ =(50 *5)/ 100= 2.5 ml