Category Archives: Stock Solution Preparation

Preparation of 0.5 M EDTA stock solution from anhydrous EDTA free acid

Preparation of 0.5 M EDTA stock solution from anhydrous EDTA

  • EDTA (EthyleneDiamineTetraAcetic acid), a polyamino carboxylic acid, is extensively used in molecular biology experiments as a chelating agent. It sequesters metal ions such as Ca2+ and Fe3+. Metal ions are necessary for the action of many enzymes including DNases.
  • EDTA is commercially available as _ _ _ _ _
    • Anhydrous EDTA (CAS Number 60-00-4, Molecular Weight 292.24)
    • Disodium EDTA dihydrate (EDTA.Na2.2H2O, CAS Number 6381-92-6, Molecular Weight 372.24) and
    • Tetrasodium EDTA tetrahydrate (EDTA.Na4.4H2O, CAS Number 13235-36-4, Molecular Weight 452.23).
  • Anhydrous free acid EDTA is least soluble among all EDTA forms in water. To dissolve anhydrous free acid EDTA in water, a lot of NaOH (3.1 ratio) is added to bring the pH to 8.0.
  • Disodium EDTA dihydrate have better solubility than anhydrous free acid EDTA and is most commonly used for the preparation of 0.5 M EDTA solution.
  • Tetrasodium EDTA tetrahydrate is soluble in water. The resulting solution has pH above 10.0, therefore is not suitable for cell and molecular biology experiments.
  • Here we show preparation of 0.5M EDTA solution from Anhydrous free acid EDTA.

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Requirements

  • Reagents
    • EDTA
    • NaOH pallet / 10N NaOH solution (for pH adjustment)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

Composition:

0.5 M EDTA, pH 8.0 at 25°C

Objective:  

Preparation of 1000 ml of 0.5 M EDTA solution, pH 8.0 in water from Anhydrous free acid EDTA (Molecular Weight 292.24)

Preparation

Step 1: Weigh out 146.12 grams EDTA.Na2.2H2O (Molecular Weight 292.24). Transfer to 2 L beaker / conical flask. Add 700 ml deionized / Milli-Q water.

Precaution: Do not dissolve in 1000 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.

Step 2: While stirring vigorously on a magnetic stirrer, add NaOH pellet to adjust the solution pH 8.0.

Tips:
  • ~40 g NaOH pellet is required to adjust the pH 8.0.
  • It is not easy to dissolve EDTA. To dissolve the EDTA completely, solution pH 8.0 is required.

Step 3:  Adjust the volume to 1000 ml with deionized / Milli-Q water. Mix it again.

Step 4:  Transfer the solution to autoclavable bottle. Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle)

Tip:
  • Depending on the consumption, one can make small aliquots of solution.
Note:
  • One can sterilize the solution by passing through 0.22 μ filter unit. Filter sterilization removes all suspended particles with size more than 0.22 μ which includes most bacteria their spores but not mycoplasma. However, it does not inactivate enzyme activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.

Storage:

Solution can be stored at 15 – 25 °C (room temperature) for several months.

Applications:

0.5 M EDTA solution is used for the preparation of many solutions including TAE, TBE, DNA loading dye, resuspension buffer (isolation of plasmid), Tris-EDTA, Trypsin-EDTA, etc.

Follow the table to prepare EDTA solution of specific concentration and volume from anhydrous EDTA Acid free (Molecular Weight 292.24).
Conc. / Volume 100 ml 250 ml 500 ml 1000 ml
10 mM 0.29 g 0.73 g 1.46 g 2.92 g
100 mM 2.92 g 7.306 g 14.612 g 29.224 g
0.25 M 7.306 g 18.265 g 36.53 g 73.06 g
0.5 M 14.612 g 36.53 g 73.06 g 146.12 g

 

Molarity

Molarity

Molarity is the most widely used unit to express solute concentration in a solution. Molarity refers to moles of solutes per litre of solution.

The molarity of a solution can be calculated as follows:

A solution which contains 1 mole of solute per litre of solution is called 1 molar.

A mole of a substance contains 6.02 x 1023 (Avogadro constant) molecules of that substance. 

One mole is equal to the molecular weight of the substance in grams.

                                                     1 mole = molecular weight of substance

For example, 40 gram of NaOH (Molecular weight = 40) is equal to 1 mole of NaOH.

Mole is calculated by dividing the total amount of substance (weight in gram) by molecular weight of that substance.
So if a solution contains 40 gram (1 mole) of NaOH per litre it is said to be 1 molar NaOH solution.

Preparing solution of specific concentration using simple dilution

Preparing solution of specific concentration using simple dilution

If you have a concentrated stock solution of known concentration, you can prepare working solution of specific concentration and volume by diluting stock solution.

Use following formula to calculate the volume of stock solution required to prepare diluted solution

                                    Cf x Vf = CS x VS

Cf: Concentration of final solution
Vf: Volume of the final solution
CS: Conc. of stock solution

VS: Volume of stock solution

Mole fraction

Mole fraction

Mole fraction refers to the fraction of a component in a solution when the amounts (component and solution) are expressed in moles. In other words, Mole fraction of a component is the ratio of moles of that particular component to the total moles of the solution.

Mole fraction (𝒳i) can be calculated by
ni = Mole of ith component of the solution

ntot = Mole of the solution

Suppose a solution contains 3 components (two solutes, A and B, and a solvent C)

Mole fraction of component A (𝒳A) =
Mole fraction of component B (𝒳B) =
Mole fraction of component C (𝒳C) =
Where
nA = Mole of component A
nB = Mole of component B

nC = Mole of component C

Since the solution contains only three components, two solutes and a solvent, the total moles (ntot) of the solution will be equal to

ntot =  nA + nB + nC

Therefore above equation can also be written

Mole fraction of component A (𝒳A)
Mole fraction of component B (𝒳B)
Mole fraction of component C (𝒳C)
Mole fraction is dimensionless:

Since value of mole fraction is obtained by dividing mass of a component by total mass of solution, it dimensionless. However, sometimes it is written as mole/mole.

The sum of all Mole fraction is equal to 1.
In above example

𝒳A + 𝒳B + 𝒳C = 1

Independent of temperature

In contrast to density, Mole fraction is independent of temperature i.e., if temperature changes, the value of Mole fraction does not change.

Mass fraction

Mass fraction

Mass fraction refers to the fraction of a component in a solution when the amounts (component and solution) are expressed in mass (weight). In other words, mass fraction is the ratio of the mass (or weight) of a component to the total mass (or weight) of the solution.

Mass fraction (ωi) can be calculated by
Where
mi = Mass of ith component of the solution

mtot = Mass of the solution

Suppose a solution is prepared by dissolving two solutes A and B in a solvent C.

Mass fraction of component A (ωA)
Mass fraction of component B (ωB)
Mass fraction of component C (ωC)
Where
mA = Mass of component A
mB = Mass of component B

mC = Mass of component C

Since the solution contains only three components, two solutes and a solvent, the total mass of the solution (mtot) will be equal to

                                                                     mtot =  mA + mB + mC

Therefore above equation can also be written
Mass fraction of component A (ωA)
Mass fraction of component B (ωB)
Mass fraction of component C (ωC)
Mass fraction is dimensionless:

Since value of mass fraction is obtained by dividing mass of a component by total mass of the solution, it dimensionless. However, sometimes it is written as gram/gram or Kg/Kg.

The sum of all mass fraction is equal to 1.
In above example

ωA + ωB + ωC = 1

Independent of temperature

In contrast to density, mass fraction is independent of temperature i.e., if temperature changes, the value of mass fraction does not change.

Solution

  • A homogeneous mixture of two or more non-reactive substances is called solution. Solution is prepared by dissolving one or more substances in another substance.
  • Solute and solvent are the two components of the solution. The dissolved component is called solute whereas component in which solute is dissolved is referred to as solvent. A solution will contain only one solvent but can contain many solutes.
  • Physical properties such as density and refractive index are uniform throughout the solution. However, these properties can vary depending on solvent and solute ratio. For example, physical properties of a 10 % (w/w) sugar solution will be different from 20% (w/w) sugar solution.

Table : Molarity of commercially supplied concentrated acids and bases

Table – Molarity of commercially supplied concentrated acids and bases

Acids and bases
Molecular formula
Molecular weight
Strength of Concentrated reagent as supplied commercially
Specific gravity (g/ml) at 25 °C
Molarity
(Mole/L)
Related Links
Formic acid
HCOOH
46.03
95 % (w/w)
1.22
Acetic acid (glacial)
CH3COOH
60.05
99.6% (w/w)
1.05
Hydrochloric acid
HCl
36.46
36 % (w/w)
1.176
11.61
37 % (w/w)
1.2
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Nitric acid
HNO3
63.01
70 % (w/w)
1.413
Phosphoric acid
H3PO4
98.00
85 % (w/w)
1.685
Sulfuric acid
H2SO4
98.07
95 % (w/w)
1.84
Ammonium hydroxide
(also known as aqueous solution of ammonia)
NH4OH
(NH3 + H2O)
35.0
56.6% (w/w)  NH4OH*
Or
28% NH3 in water
0.90
Potassium Hydroxide
KOH
 56.11
45.0% (w/w)
1.456
Sodium Hydroxide
NaOH
40.0
50.0% (w/w)
1.515

* 56.6% Ammonium hydroxide solution contains 28% (w/w) ammonia (NH3). Ammonia is a gas which dissolves readily in water. In aqueous solution, it is present as Ammonium hydroxide.

Preparation of 1N hydrochloric acid (HCl)

Overview

  • HCl is supplied as 36 -38% (w/w) concentrated liquid form.
  • The normality of a 37% HCl solution  (average conc.) which has density 1.2 g/ml, is 12.18 N.

Requirements

  • Reagents
    • 37% (w/w) stock solution of HCl
    • Deionized/Double distilled water
  • Equipment and disposables
    • Measuring cylinder
    • Beaker
Objective:

Preparation of 100 ml of 1N HCl solution from stock solution of HCl [37% (w/w)]

Procedure:

Step 1: Calculate the volume of conc. stock solution required to prepare 1N HCl solution

Use formula: Cf * Vf = CS * VS

Cf: Concentration of final solution = 1 N
Vf: Volume of the final solution = 100 ml
CS: Concentration of stock solution = 12.18 N

VS: Amount stock solution required i.e., Unknown

                               1 X 100 = 12.178 X VS           OR

                               VS = 1 X 100 / 12.18      =    8.2 ml

                              Amount of water = 100 – 8.2 = 91.8 ml

Step 2:  Take 91.8 ml water in a measuring cylinder and add 8.2 ml concentrated HCl. Mix. Transfer solution to a appropriate glass bottle.

Storage: Solution can be stored at room temperature for several months.

Application: 1N HCl can be used for pH adjustment.

Preparation of 10 M Sodium hydroxide (NaOH) solution

Overview:

A 10N sodium hydroxide stock solution is prepared which is used for many applications including including adjusting pH of various solutions.

Requirements

  • Reagents
    • NaOH pallet
    • Deionized / Double distilled water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

Composition: 10 M NaOH solution

Objective: Preparation of 100 ml of 10 M NaOH solution in water

Preparation

Step 1: To prepare 100 ml of 10 M NaOH solution, weigh out 40 g of NaOH (Mol. Wt. 40).

Precaution: Don’t add water in the NaOH pellet. Instead add NaOH pellet in the water (few pellet at a time)

Step 2: Dissolve NaOH pellet in 70 ml water by adding small amount of NaOH pellet at a time.
  • Take 70 ml deionized/double distilled water in a beaker.
  • Add few pellet (4 – 5 pellet each time) of NaOH in water and wait until they dissolve. Add few more pellet.

Precaution: The preparation of 10 N NaOH is a highly exothermic reaction, which can cause breakage of glass beaker. One can keep the beaker on ice and can add NaOH pellet.

Step 3: Adjust the volume to 100 ml with deionized / double distilled water.
  • Transfer solution to 100 measuring cylinder and adjust the volume to 100 with deionized/ double distilled water.

Note: There is no need to sterilize this solution by autoclaving. No microorganism will grow in concentrated NaOH solution.

Storage:

Solution can be stored at room temperature for several months.

Applications

  • NaOH is a strong base and commonly used to adjust the pH of many solution.  
  • Plasmid isolation by alkaline lysis method
  • Preparation of EDTA solution
Follow the table to prepare NaOH solution of specific concentration and volume or use calculator
Conc. / Volume 50 ml 100 ml 250 ml 500 ml
1 M 2 g 4 g 10 g 20 g
2 M 4 g 8 g 20 g 40 g
5 M 10 g 20 g 50 g 100 g
10 M 20 g 40 g 100 g 200 g
Abbreviations:
g = gram
Conc. = Concentration

Mol. Wt. = Molecular weight

Preparation of Ethidium Bromide stock solution (10 mg/ml)

Overview:

  • Ethidium bromide (EtBr) is a commonly used fluorescent stain to visualize nucleic acid especially DNA in agarose gels.
  • Ethidium bromide intercalates between DNA bases. Upon intercalation, its fluorescence increases several folds (25 fold increase when it binds DNA), much higher than the unbound ethidium bromide fluorescence, therefore, eliminates the need to wash gel to remove unbound ethidium bromide.
  • When exposed to uv light, it appears bright pink/orange colour.
  • Ethidium bromide can detect as little as 1 ng DNA/band in agarose gel.
  • It can also bind to RNA which results in 21 fold increase in its fluorescence intensity.
  • It has been reported to bind to single-stranded DNA.
  • Ethidium bromide powder is quite stable at room temperature but need to be protected from exposure of light.

Requirements

  • Reagents
    • Ethidium bromide
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask/Beaker/15-ml screw-cap graduated polypropylene centrifuge tube
    • Magnetic stirrer / Tube-Rotator

Composition

  • 10 mg/ ml ethidium bromide in water

Objective

  • Preparation of 10 ml of 10 mg/ml ethidium bromide solution in water

Preparation

General precaution:

Wear gloves and lab coat at all times when handling ethidium bromide or ethidium bromide contaminated solutions, glassware, pipette tips, and so forth.

Step 1: Weigh out 100 mg ethidium bromide conical flask / beaker / 15-ml polypropylene centrifuge tube. Add 7 – 8 ml water.

Tip:
  • We recommend you to use 15-ml screw-cap graduated polypropylene centrifuge tube. Since these tubes have milliliter marks, you can adjust the solution volume without transferring the solution to measuring cylinder. Moreover, you don’t need to contaminate reusable vessels (beaker).

Step 2: Mix until all ethidium bromide dissolves completely. This may take long time.

Tip:
  • Use tube rotator to mix all content if you 15-ml screw-cap graduated polypropylene centrifuge tube is used.
Precaution:
  • Cover the tube with aluminium foil to protect ethidium Bromide from light exposure.

Step 3: Adjust the volume to 10 ml with Deionized / Milli-Q water.

Note:
  • The solution will appear red.

Ethidium bromide solution is ready for use.

Storage:

  • Ethidium bromide is quite stable and can be stored at 4°C for many years if protected from light.

Application

  • Detection of DNA and RNA on agarose gel.
  • Purification of supercoiled DNA using cesium chloride – ethidium bromide gradient centrifugation method