Question

BUFFERS:

(Final concentration for 1x is 20 mM Tris; 500 mM NaCl; 1 mM CaCl₂; 1 mM MgCl₂; pH 7.4)

5x ConA Buffer, (1L)

    •       700 ml dH₂O, set stirring and add:

    •       100 ml 1M Tris, pH 7.2-7.4

    •       5 ml 1M MgCl₂

    •       5 ml 1M CaCl₂

    •       146 g NaCl

    •       Check pH is between 7.2 and 7.4

    •       To 1000 ml with dH₂O

Elution Buffer A: ~0.2M AMM: 0.1 g alpha methyl mannopyranoside in 2 mls ConA buffer

Elution Buffer B: ~0.5M AMM: 0.2 g alpha methyl mannopyranoside in 2 mls ConA buffer

Elution Buffer C: ~1 M AMM: Add 0.4 g alpha methyl mannopyranoside in 2 mls ConA buffer (you may need more of this)

Formula weight for alpha methyl mannopyranoside is 194.2 g.

Questions:

Buffers: Calculate the concentrations of Tris, CaCl2 and MgCl2 in the 5x ConA Buffer stock

Tris:

CaCl2:

MgCl2:

For Buffer B: What is the exact concentration of the AMM to three significant figures

Answer 1

Buffers: Formula for calculating concentration is C₁ V₁ =C₂ V₂

Here C₁ andV₁ are initial concentration and volume, while C₂ andV₂ are final concentration and volume of any specific compount being diluted.

a. Tris - 100 ml (V₁), 1M tris (C₁) is being diluted to an unknown concentration (C₂) with a total volume of 1000 ml (V₂). now appying the formula C₂ = (C₁ * V₁)/V₂ = (1M * 100 ml)/ 1000 ml = 0.1 M

Tris = 0.1 M

b. CaCl₂ - 5 ml (V₁), 1M CaCl₂ (C₁) is being diluted to an unknown concentration (C₂) with a total volume of 1000 ml (V₂). now appying the formula C₂ = (C₁ * V₁)/V₂ = (1M * 5 ml)/ 1000 ml = 0.005 M

CaCl₂ = 0.005 M (or 5 mM)

c. MgCl₂ - 5 ml (V₁), 1M MgCl₂ (C₁) is being diluted to an unknown concentration (C₂) with a total volume of 1000 ml (V₂). now appying the formula C₂ = (C₁ * V₁)/V₂ = (1M * 5 ml)/ 1000 ml = 0.005 M

MgCl₂ = 0.005 M (or 5 mM)

For Buffer B:

Molarity is number of moles per liter. 0.2 g of AMM is being dissolved in 2 ml. Converting this gives 0.2*500 g in 2 ml * 500 (gives 1 liter) which gives 100g in 1 liter.

Number of moles = mass/molecular weight = 100/194.2 = 0.514933 M

Concentration of AMM = 0.515 M

Note: If this 5x ConA buffer were to be diluted 5 times, the calculated concentrations of tris, MgCl₂ and CaCl₂ (upon division by 5) gives the same values as mentioned in first line in bracets " Final concentration for 1x is 20 mM Tris; 500 mM NaCl; 1 mM CaCl₂; 1 mM MgCl₂; pH 7.4"