# Efficient Buffer Preparation: 500ml MgCl2, Glucose, ATP, and Tris-Cl at pH 7.8

• vande060
In summary, the conversation discussed the preparation of a 500ml reaction buffer with specific concentrations of MgCl2, glucose, ATP, and Tris-Cl. The necessary calculations were made using the formulas C1V1=C2V2 and the given molar mass of each component. It was suggested to prepare a new buffer using the Henderson-Hasselbalch equation for the Tris component.
vande060

## Homework Statement

Prepare 500ml of a reaction buffer containing .82% MgCl2, 62.5 mM glucose, 950 ATP, and 100mM Tris -Cl, pH 7.8 Use the stock solutions you have prepared

we had to prepare in previous problems:

500ml 1.75M glucose
25mL 2.5% glucose
2L of 1.8 Tris, pH 8.5

## Homework Equations

C1V1 = C2V2
Pka Tris = 8.1
glucose mol mass = 180.16
ATP mol mass = 507.8

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## The Attempt at a Solution

my calculation for MgCl2:

.82% = .82g/100ml = 8.2g/L
15% = 15g/100ml = 150g/L

C1V1=C2V2

150*x = 8.2*500

x = 28mL stck 15% MgCl2

My calculation for glucose

1.75M glucose = 315g/L
62.5mM = 11.2 g/L

C1V1=C2V2

11.2*500 = 315*x

x = 17.7 ml 1.75M glucose stock

Here is my calculation for ATP

5mM ATP = 3g/L
950nM ATP = .00048g/L

C1V1=C2V2

3*x = .00048*500

x =.08ml of stocK 5mM ATP

My calculation for the Tris I am not sure about, can I use my original pH 8.5 solution the prof asked me to prepare, or do I have to start over with the Tris acid and base components and use henderson H equation? does this all look right so far?

C1V1 = C2V2 works for molar concentrations as well, you don't have to convert them to g/L.

My guess is you should prepare new buffer from HH equation.

Borek said:
C1V1 = C2V2 works for molar concentrations as well, you don't have to convert them to g/L.

My guess is you should prepare new buffer from HH equation.

is the rest of it right?

Looks OK, but I just skimmed.

First of all, great job on your calculations for MgCl2, glucose, and ATP! Your calculations seem to be correct and you have used the correct equations to determine the volumes of stock solutions needed.

For the Tris component, you can use the original pH 8.5 solution that you prepared in a previous problem. However, you will need to adjust the pH to 7.8 by using the Henderson-Hasselbalch equation. The pKa of Tris is 8.1, so you can use the equation pH = pKa + log([base]/[acid]) to determine the amount of Tris acid and base needed to achieve a pH of 7.8. Once you have adjusted the pH, you can use the same equation (C1V1 = C2V2) to determine the volume of stock solution needed.

Overall, your approach and calculations seem to be correct. Just make sure to double check your calculations and units before preparing the buffer. Good luck!

## 1. How do I prepare a 500ml solution of MgCl2, Glucose, ATP, and Tris-Cl at pH 7.8 efficiently?

To prepare this solution efficiently, first calculate the amount of each reagent needed based on the desired final concentration. Then, add the reagents in the following order: MgCl2, glucose, ATP, and Tris-Cl, while stirring continuously to ensure they are completely dissolved. Finally, adjust the pH to 7.8 using a pH meter or indicator and add water to bring the total volume to 500ml.

## 2. What is the purpose of each component in this buffer solution?

MgCl2 is a divalent cation that helps stabilize enzymes and proteins. Glucose is a source of energy for cells. ATP is a high-energy molecule that serves as a substrate for many biochemical reactions. Tris-Cl is a buffering agent that helps maintain the solution at a constant pH.

## 3. Can I use a different buffer system instead of Tris-Cl at pH 7.8?

Yes, you can use a different buffer system as long as it is compatible with your experimental conditions and maintains a pH of 7.8. Some alternative buffer systems include HEPES, MES, and MOPS.

## 4. How should I store this buffer solution?

This buffer solution should be stored at 4°C when not in use. It is recommended to make fresh solution every 1-2 weeks to ensure maximum efficiency and avoid contamination.

## 5. What precautions should I take while preparing this buffer solution?

It is important to accurately measure the amount of each reagent and to add them in the correct order to ensure proper dissolution. It is also important to use high-quality water, such as deionized or distilled water, to avoid any impurities in the solution. Additionally, wear appropriate personal protective equipment, such as gloves and goggles, to avoid any contact with the reagents.