Monthly Archives: April 2017

Preparation of 10% (w/v) Sodium dodecyl sulfate (SDS) solution

Overview

  • Sodium dodecyl sulfate (SDS), also known as Sodium lauryl sulfate (SLS), is an anionic surfactant (detergent).
  • It is extensively used in biological research as a strong protein denaturant and lipid-solubilizing agent.
  • It is also used in our cleaning and personal care products, e.g., shampoos, hand soaps, bath products, shaving creams, toothpaste.
  • SDS is water soluble, white to yellowish powder.
  • Heating dissolves SDS quickly in water, thus helpful to prepare concentrated SDS aqueous solution (20% SDS solution in water). Once dissolved, SDS forms clear transparent or slight yellowish solution.
  • 10% or 20% (w/v) stock solution of Sodium dodecyl sulfate (SDS) is generally prepared in distilled water (or milli Q water).

Requirement

  • Reagents
    • Sodium dodecyl sulfate (CH3(CH2)11OSO3Na, Molecular weight: 288.38, CAS Number: 151-21-3)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer with heat control (optional)

Composition

  • 10 % Sodium dodecyl sulfate

Objective

  • Preparation of 100 ml of 10 % SDS in Deionized / Milli-Q water.

Preparation

Step 1: To prepare 100 ml of 10 % SDS solution, weigh out 10 grams SDS in 250 ml conical flask / beaker. Add 80 ml deionized / Milli-Q water and mix it.

Tips:

  • One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.

Precautions:

  • Do not dissolve in 100 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute is dissolved in solvent.
  • Wear face mask to avoid inhalation of SDS dust.
  • Try to avoid frothing.

Step 2: Heat to 68 °C if necessary.

Tips:

  • One can set the magnetic stirrer temperature 68 °C. If magnetic stirrer is unavailable, place the solution in a water bath set at 68 °C.

Precautions:

  • Make sure to dissolve any residual powder sticking to the glass.

Step 3: Adjust the volume to 100 ml with deionized / Milli-Q water. Mix it again.

Step 4 (Optional) : One can filter the solution to remove any undissolved material.

Notes:

  • Do not autoclave 10% SDS solution.

Storage

  • Solution can be stored at room temperature for several months.

Precautions:

  • Do not store the solution at 4°C. SDS will precipitate at temperature below 15°C. If there is any precipitate, redissolve by warming the solution (68°C for 10 min)

Applications

  • Plasmid isolation by alkaline lysis method
  • SDS-PAGE
  • Preparation of Laemmli sample buffer
  • Stripping of immunoblot
Follow the table to prepare Sodium dodecyl sulfate (SDS) solution of various volume (10 ml, 100 ml, 500 ml and 1,000 ml) and percentage (1%, 5%, 10% and 20%).
Reagents / Volume 10 ml 100 ml 500 ml 1000 ml
1% 0.1 g 1.00 g 5.00 g 10.00 g
5% 0.50 g 5.00 g 25.00 g 50.00 g
10% 1.00 g 10.00 g 50.00 g 100.00 g
20% 2.00 g 20.00 g 100.00 g

200.00 g

 

Preparation of stock solution of ampicillin

Overview

  • Ampicillin belongs to a group of beta-lactam antibiotics.
  • Ampicillin is used to treat many different types of infections caused by bacteria, e.g., bladder infections, ear infections, gonorrhea, pneumonia and E. coli or salmonella infection.
  • Ampicillin is one of the synthetic derivatives of penicillin. It was generated by adding an amino group side chain on the penicillin.
  • In contrast to penicillin which is active only against gram-positive bacteria, ampicillin is active against both gram-positive and gram negative bacteria.
  • Ampicillin inhibits the synthesis of cell wall of bacteria by stopping the peptidoglycan cross-linking.
  • Ampicillin functions as a competitive inhibitor of the enzyme transpeptidase which is required for the synthesis of bacterial cell wall.
  • Due to inhibition of cell wall synthesis, bacteria do not grow and have risk to die due to lysis in the presence of ampicillin. However, as the ampicillin concentration decreases below critical level in the surroundings, live bacteria resume the cell wall synthesis and growth.
  • Ampicillin is one of the most extensively used as selection marker in molecular biology and bacteriology. Many commonly used vectors have ampicillin resistance gene.
  • 50 – 100 mg/ml stock solution of ampicillin is generally prepared in water. Depending on the need, stock solution is diluted to final concentration.

Requirement

  • Reagents
    • Ampicillin Sodium salt
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • 15 ml screw-capped tube / Conical flask / Beaker / 15 ml screw-capped tube

Composition

  • 50 mg/ml ampicillin

Objective

  • Preparation of 10 ml of 50 mg/ml ampicillin stock solution in water

Preparation

Step 1: To prepare 10 ml of 50 mg/ml stock solution of ampicillin, weigh out 500 mg of ampicillin. Transfer it to 15 ml screw-capped tube / Conical flask / Beaker. Add 9 ml of deionized / Milli-Q water. Mix until all ampicillin is completely dissolved.

Tips:

  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml deionized / Milli-Q water and mix the content by inverting the tube number of times or using rotator. Once the content is mixed, make up the volume 10 ml. Since the tube is marked, you don’t need to transfer the content to measuring cylinder.
  • Use autoclaved sterile deionized / Milli-Q water.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

Step 3: Filter sterilize by passing the solution through 0.2 micron filter paper under aseptic condition.

Storage

  • Store the solution at -20°C for long time.

Tips:

  • It is recommended to store the ampicillin stock solution as small aliquots (1 ml). Repeated freeze-thaw cycle can cause ampicillin degradation.
  • Aliquoting must be done under sterile condition.

Applications

  • A selection marker for bacterial culture during gene cloning experiments.
Follow the table to prepare ampicillin solution of different concentration and volume.
Conc. / Volume 5 ml 10 ml 25 ml 100 ml
50 mg / ml 0.25 g 0.5 g 2.5 g 5 g
100 mg / ml 0.5 g 1 g 5 g

10 g

 

Preparation of stock solution of chloramphenicol

Overview

  • Chloramphenicol is a broad spectrum antibiotics which is effective against both gram positive and gram negative bacteria.
  • It is bacteriostatic agent.
  • It inhibits bacterial protein synthesis by irreversibly binding to 50S ribosomal subunit and inhibiting peptidyl transferase catalytic function.

Requirement

  • Reagents
    • Chloramphenicol
    • 70% ethanol
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer

Composition

  • 34 mg/ml chloramphenicol
  • 70% ethanol

Objective

Preparation of 10 ml of chloramphenicol stock solution in 70% ethanol

Preparation

Step 1: To prepare 10 ml of stock solution of chloramphenicol, weigh out 340 mg of chloramphenicol. Transfer it to 15 ml screw-capped tube. Add 9 ml of 70% ethanol. Mix until all chloramphenicol is dissolved completely.

Tips:

  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml 70% ethanol and mix the content by rotating the tube. Once the content is mixed, make up the volume 10 ml.

Precautions:

  • Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.

Step 2: Adjust the volume to 10 ml with d70% ethanol. Mix it again.

Storage

Store the solution in small aliquots (1 ml) at -20°C for long time.

Tips:

  • It is recommended to store the chloramphenicol stock solution in small aliquots.

Applications

A selection marker for bacterial culture during gene cloning experiments.

Follow the table to prepare chloramphenicol solution of various volume (5 ml, 10 ml, 25 ml, 100).
Reagents / Volume 5 ml 10 ml 25 ml 100 ml
Chloramphenicol 170 mg 340 mg 850 mg 3400 mg
70% Ethanol Adjust the final volume to 5 ml Adjust the final volume to 10 ml Adjust the final volume to 25 ml Adjust the final volume to 100 ml

Preparation of stock solution of Kanamycin

Overview

  • Kanamycin is a water-soluble, aminoglycoside bactericidal antibiotic.
  • It was originally purified from the bacterium Streptomyces kanamyceticus.
  • It inhibits protein synthesis in prokaryotes by binding to the 30S subunit of the bacterial ribosomes.
  • Kanamycin is effective against a wide variety of gram-negative and some gram-positive bacteria.

Requirement

  • Reagents
    • Kanamycin
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer

Composition

  • 50 mg/ml kanamycin

Objective

  • Preparation of 10 ml of 50 mg/ml kanamycin stock solution in water

Preparation

Step 1: To prepare 10 ml of stock solution of kanamycin, weigh out 500 mg of kanamycin. Transfer it to 15 ml screw-capped tube. Add 9 ml of deionized / Milli-Q water. Mix until all kanamycin is completely dissolved.

Tips:
  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml deionized / Milli-Q water and mix the content by rotating the tube. Once the content is mixed, make up the volume 10 ml.
Precautions:
  • Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.
  • Use autoclaved deionized / Milli-Q water.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

Storage

  • Store the solution in small aliquots (1 ml) at -20°C for long time.

Applications

  • A selection marker for bacterial culture in gene cloning experiments.
Follow the table to prepare kanamycin solution of different concentration and volume.
Conc. / Volume 5 ml 10 ml 25 ml 100 ml
50 mg / ml 0.25 g 0.5 g 2.5 g 5 g
100 mg / ml 0.5 g 1 g 5 g 10 g

Preparation of stock solution of streptomycin

Overview

  • Streptomycin function by inhibiting protein synthesis in bacteria.
  • Binding of streptomycin to the small 16S rRNA of the 30S subunit of the bacterial ribosome subunit prevent access of aminoacyl tRNA to the acceptor site on the mRNA – ribosome complex, leading to inhibition of protein synthesis.
  • It binds to the small 16S rRNA of the 30S subunit of the bacterial ribosome, interfering with the binding of formylmethionyl-tRNA to the 30S subunit.

Requirement

  • Reagents
    • streptomycin
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer

Composition

  • 50 mg/ml streptomycin

Objective

  • Preparation of 10 ml of 50 mg/ml streptomycin stock solution in water

Preparation

Step 1: To prepare 10 ml of stock solution of streptomycin, weigh out 100 mg of streptomycin. Transfer it to 15 ml screw-capped tube ml / beaker / conical flask. Add 9 ml of deionized / Milli-Q water. Mix until all streptomycin is dissolved completely.

Tips:

  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml deionized / Milli-Q water and mix the content by rotating the tube. Once the content is mixed, make up the volume 10 ml.

Precautions:

  • Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.

Step 2: Adjust the volume to 10 ml with deionized / Milli-Q water. Mix it again.

Storage

  • Store the solution in small aliquots (1 ml) at -20°C for long time.

Note:

  • It is recommended to store the tetracycline stock solution in small aliquots.

Applications

  • A selection marker for bacterial culture during gene cloning experiments.

 

Follow the table to prepare streptomycin solution of various volume (5 ml, 10 ml, 25 ml, 100).
Reagents / Volume 5 ml 10 ml 25 ml 100 ml
streptomycin 50 mg 100 mg 250 mg 1000 mg
Deionized / Milli-Q water Adjust the final volume to 5 ml Adjust the final volume to 10 ml Adjust the final volume to 25 ml Adjust the final volume to 100 ml

Preparation of stock solution of tetracycline

Overview

  • Tetracyclines are a group of broad-spectrum antibiotics. They are active against wide range of microorganisms including bacteria (both gram positive and gram negative), chlamydiae, mycoplasmas, rickettsiae, and protozoan parasites.
  • Tetracyclines function by inhibiting the protein synthesis.
  • Binding of tetracyclines to 30S ribosomal subunit prevent access of aminoacyl tRNA to the acceptor site on the mRNA – ribosome complex, leading to inhibition of protein synthesis.
  • Tetracycline hydrochloride can be used for biological research.
  • Tetracycline hydrochloride is soluble in water (50 mg/ml) with heating.
  • It forms a clear, yellow to yellow-orange solution in water.
  • It also forms a clear, yellow-green solution in 95% ethanol.
  • Tetracycline is rapidly destroyed by alkali hydroxide solutions and solutions in water become turbid on standing due to hydrolysis and precipitation of tetracycline.
  • The potency of tetracycline is reduced in solutions with pH below 2.
  • A 5 – 12 mg/ml stock solution of tetracycline is generally prepared in 70% ethanol. Stock solution is diluted to final concentration, depending on the need.
  • Magnesium ions are antagonistic to tetracycline, therefore, don’t use magnesium salt in bacterial culture medium if tetracycline is going to be used for selection. This may lead to false positives.

Requirement

  • Reagents
    • Tetracycline hydrochloride (C2H24N2O8.HCl) (Molecular weight: 480.9)
    • 70% ethanol
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker / 15 ml screw-capped tube
    • Magnetic stirrer

Composition

  • 12 mg/ml tetracycline
  • 70% ethanol

Objective

  • Preparation of 10 ml of tetracycline stock solution (12 mg/ml) in 70% ethanol

Preparation

Step 1: To prepare 10 ml of 12 mg/ml tetracycline stock solution, weigh out 120 mg of tetracycline. Transfer it to 15 ml screw-capped tube. Add 9 ml of 70% ethanol. Mix until all tetracycline dissolves completely.

Tips:

  • We recommend to use 15 ml screw-capped tube with millilitre marks. Transfer all the contents in it. Add 9 ml 70% ethanol and mix the content by rotating the tube. Once the content is mixed, make up the volume 10 ml.

Precautions:

  • Do not dissolve in 10 ml of deionized / Milli-Q water. In most cases, solution volume increases when the large amount of solute dissolves in solvent.

Step 2: Adjust the volume to 10 ml with 70% ethanol. Mix it again.

Storage

Store the solution in small aliquots (1 ml) at -20°C for long time.

Precautions:

  • Protect the solution from light.

Applications

  • A selection marker for bacterial culture during gene cloning experiments.
  • Often used for inducible expression system (tet-on tet-off) in mammalian cells.
Follow the table to prepare tetracycline stock solution of various volume (5 ml, 10 ml, 25 ml, 100) and concentration.
Conc. / Volume 5 ml 10 ml 25 ml 100 ml
5 mg/ml 25 mg 50 mg 125 mg 500 mg
10 mg/ml 50 mg 100 mg 250 mg 1000 mg
12 mg/ml 60 mg 120 mg 300 mg

1200 mg

Stock solution

Overview :

  • Stock solution can be defined as a highly concentrated solution, mostly prepared as 10X concentrated, which can be diluted to prepare working solution.
  • Stock solution is also used as a component to prepare complex solution containing many ingredients. For example, stock solution of EDTA (0.5 M EDTA solution) can be used to prepare many solutions including TAE, TBE, Tris-EDTA, Trypsin-EDTA etc.

Advantages :

  • Since stock solution is highly concentrated, its storage requires less space.
  • It is also convenient to transport stock solution due to its small volume.
  • The concentration of various component of working solution is more accurate when it is made from stock solution rather than individual component from its original form.
  • Stock solutions which contains any active biological substances (e.g., enzymes, inhibitors, DNA, RNA etc.) are more stable in highly concentrated form.

Disadvantages :

  • Since stock solution is highly concentrated, often to dissolve them requires more time and effort. Sometime, heating of solution is required to dissolve the substance completely.
  • Long term storage of stock solution (especially salt solution, at cold room) may leads to precipitation of solute.

Preparation of 3 M Sodium acetate (CH3COONa) solution, pH 5.2

Overview

  • Sodium acetate is a sodium salt of acetic acid.
  • It is water-soluble.
  • Anhydrous sodium acetate is very hygroscopic. It readily converts to trihydrate due to its hygroscopic nature.

Related Content

Requirements

  • Reagents
    • Sodium acetate, anhydrous (CH3COONa) (Molecular weight = 82.0343)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer

Composition

  • 3 M Sodium acetate

Objective

Preparation of 100 ml of 3M Sodium acetate solution, pH 5.2 in water from anhydrous sodium acetate (CH3COONa)

Preparation

Step 1: To prepare 100 ml aqueous solution of 3M Sodium acetate (CH3COONa), weigh out 24.61 gram CH3COONa (Molecular weight = 82.0343). Transfer to 250 ml beaker/conical flask. Add 80 ml deionized / Milli-Q water. Mix until sodium acetate is completely dissolved.

Tip:
  • One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the dissolving process easy and convenient.
Precaution:
  • Do not dissolve in 100 ml of deionized / Milli-Q water. In most cases, solution volume increases when a large amount of solute dissolves in the solvent.

Step 2: Adjust the pH to 5.2 with glacial acetic acid.

Precaution:
  • Since pH is dependent on temperature, one should adjust pH at room temperature (25°C).

Step 3: Adjust the volume to 100 ml with deionized / Milli-Q water. Mix it again.

Note:
  • The solution will appear colorless and transparent.

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 the solution.
Note:
  • One can sterilize a solution by passing through 0.2μ filter unit. Filter sterilization removes all suspended particles with size more than 0.2 μ which includes most bacteria and their spores but not mycoplasma. Moreover, 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 room temperature.

Applications

  • Precipitation of nucleic acid (DNA and RNA)
  • Buffer solution
  • Protein crystallization
Follow the table to prepare Sodium acetate (CH3COONa) solution of different concentration and volume.
Conc. / Volume 10 ml 50 ml 100 ml 250 ml 500 ml 1000 ml
0.1 M 0.08 g 0.41 g 0.82 g 2.05 g 4.10 g 8.20 g
0.5 M 0.41 g 2.05 g 4.10 g 10.25 g 20.51 g 41.01 g
1 M 0.82 g 4.10 g 8.20 g 20.51 g 41.01 g 82.03 g
3 M 2.46 g 12.31 g 24.61 g 61.52 g 123.045 g 246.09 g

Preparation of 10X Tris-EDTA (TE) solution

Overview

  • Tris-EDTA (pH 8.0) solution is used for dissolving and storing DNA.
  • Storing DNA in slightly alkaline condition reduces the risk of depurination.
  • Tris functions as a buffering agent, and maintains the correct pH of the solution.
  • EDTA protects nucleic acid (DNA and RNA) from modification and degradation by sequestering divalent cations. Divalent cations are required for the action of many DNA modifying enzymes and DNases.

Requirement

  • Reagents
    • 1M Tris-HCl pH 8.0
    • 0.5 M EDTA pH 8.0
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer with heat control (optional)

Composition of 10X Tris-EDTA solution

  • 100 mM Tris
  • 10 mM EDTA

Composition of 1X Tris-EDTA solution

  • 10 Trypsin
  • 1 mM EDTA

Note: To prepare 1X solution, take 1 volume of 10X solution, add 9 volume of deionized / Milli-Q water. Mix it.

Objective

  • Preparation of 100 ml of 10X Tris-EDTA solution

Preparation

Step 1: Take 88 ml deionized / Milli-Q water in 250 ml beaker/conical flask. Add 10 ml of 1M Tris.Cl (pH 8.0) and 2 ml of 0.5 M EDTA (pH 8.0). Mix it.

Tips:

  • One can use manual shaking using a glass pipette to mix the ingredients. Magnetic stirrer makes the mixing process easy and convenient.
  • It is recommended to add 1M tris and 0.5M EDTA in water instead of adding them together and then adding water.

Step 2: Check the pH. It should be 8.0. If pH is not 8.0, adjust the pH with HCl or NaOH.

Step 3: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).

Notes:

  • You don’t need to autoclave the solution if you have used all autoclaved glassware and stock solutions.
  • If dissolved DNA is to be used for transfection experiments in cell culture, make sure that your tris-EDTA solution is sterile and free of any live organisms. For this purpose, do all the preparation steps inside the laminar flow hood.
  • One can also 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 and their spores but not mycoplasma. Moreover, 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

  • 1X or 10X Tris-EDTA solution can be stored at 15 – 25 °C (room temperature).

Note: Storing concentrated (10 X) solution at cold room (4 °C) may cause precipitation of solution.

Applications

  • Tris-EDTA (pH 8.0) is often used to dissolve and store DNA.
To prepare 10X Trypsin-EDTA of various volume 10 ml, 50 ml, 100 ml, 500 and 1000 ml), follow the table.
Reagents / Volume 10 ml 50 ml 100 ml 500 ml 1000 ml
1M Tris, pH 8.0 1 ml 5 ml 10 ml 50 ml 100 ml
0.5 M EDTA, pH 8.0 0.5 ml 1 ml 2 ml 10 ml 20 ml
Deionized / Milli-Q water Adjust to 10 ml Adjust to 50 ml Adjust to 100 ml Adjust to 500 ml Adjust to 1000 ml

 

Ethylenediaminetetraacetic acid (EDTA)

  • EDTA is a polyamino carboxylic acid. It is a colourless, water soluble solid. It’s conjugate base is named ethylenediaminetetraacetate.
  • EDTA is best known for its chelating property. It sequesters metal ions such as Ca2+ and Fe3+.
  • Most applications of EDTA rely on its ability to chelate metal ions. After being bound by EDTA, metal ions remain in solution but exhibit diminished reactivity. Metal ions are necessary for the action of many enzymes including DNases.
  • In molecular biology laboratories, EDTA is used for the preparation of many solutions including TAE, TBE, DNA loading dye, resuspension buffer (isolation of plasmid), Tris-EDTA, Trypsin-EDTA, etc.
  • It has been widely used in domestic products like food preservative and cosmetic stabilizer. It is also used extensively to dissolve limescale. Our daily use products like personal care, skin care, processed foods, cosmetic preparations and cleaning products often contains EDTA. It has extensive medical, engineering, agricultural and industrial applications as well.
  • EDTA applications in medical science (chelation therapy)
    • Eye drops containing EDTA are used to treat calcium deposits in the eye.
    • EDTA is sometimes used as an ointment for skin irritations produced by metals such as chromium, nickel, and copper.
    • EDTA is also used to treat lead poisoning or heavy metal toxicity.