Preparation of 10x MOPS Electrophoresis Buffer

OVERVIEW

MOPS is a zwitterionic physiological buffer, with pH range 6.5 – 7.9. MOPS-containing  electrophoresis buffer is suitable for RNA denaturing Agarose Gel Electrophoresis.

REQUIREMENTS

Reagents and solutions
MOPS free acid (C7H15NO4S, Molecular weight: 209.26)
Sodium acetate, anhydrous (CH3COONa, Molecular weight: 82.03)
0.5M EDTA, pH 7.5
Deionized / Milli-Q water

Equipment and disposables
Measuring cylinder 
Conical flask / Beaker 
Magnetic stirrer

Note: Use ultrapure reagents and DEPC-treated water and glasswares if the MOPS Running Buffer is to be used for RNA electrophoresis.

COMPOSITION

Composition of 10X buffer
0.2 M MOPS 
0.05 M Sodium Acetate 
0.01 M EDTA
pH 7.0

Composition of 1X buffer
0.02 M MOPS 
0.005 M Sodium Acetate 
0.001 M EDTA
pH 7.0

Objective:
Preparation of 1 L MOPS running buffer

PROCEDURE

Step 1: Weigh out 41.85 g MOPS free acid (C7H15NO4S, Molecular weight: 209.26) and 4.1 g Sodium acetate, anhydrous (CH3COONa, Molecular weight: 82.03). Transfer to a 2-liter beaker / conical flask. Add 800 ml deionized / Milli-Q water.

Note
1. If you do not have 0.5M EDTA solution, you can also add 3.72 g of EDTA Disodium dihydrate (Molecular weight: 372.24). Since EDTA takes time to dissolve, it is convenient to add EDTA stock solution.
2.
You can also use MOPS sodium salt in place of MOPS free acid. You need to add 46.25 g of MOPS sodium salt.

Precaution
Do not dissolve all ingredients in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solutes dissolve in a solvent. 

Tip
It is always a good practice to dissolve solute(s) in a small volume of solvent (≈ 80% of the final solution volume). After the solute is completely dissolved and pH is adjusted, make up the correct volume with the solvent.

Step 2: Add 20 mL of 0.5M EDTA solution and mix the solution again.

Step 3: Adjust the pH to 7.0 using 10 N NaOH solution.

Step 4: Adjust the volume to 1000 ml with deionized/Milli-Q water. Mix it again.

Step 5: Sterilize the solution by filtering through a sterile filter unit containing a membrane with porosity 0.22 µm or less. 

Precaution
We don’t recommend autoclaving the solution. 

Note
Freshly prepared solution will appear clear colourless liquid which can turn into yellowish with time. 

STORAGE
Stored the solution at 4° C in a brown bottle 

Precaution
Protect the solution from light.

Follow the table to prepare a 10x MOPS electrophoresis buffer of various volumes.
Reagents / Volume100 ml250 ml500 ml1000 ml
MOPS free acid4.185 g10.46 g20.93 g41.85 g
Sodium acetate, anhydrous0.41 g1.025 g2.05 g4.1 g
0.5 M EDTA (pH 8.0)2 ml5 ml10 ml20 ml
WaterAdjust to the final volume 100 mlAdjust to the final volume 250 mlAdjust to the final volume 500 mlAdjust to the final volume 1000 ml

REFERENCES

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