Category Archives: Solution Preparation

Preparation of 1 mM EDTA solution from stock solution of EDTA (0.5M, pH 8.0)

Overview:

  • EDTA (Ethylenediaminetetraacetic acid) is a chelating agent. Due to its ability to sequester metal ions such as Mn2+, Ca2+, Mg2+ and Fe3+, EDTA is widely used in number of cell and molecular biology experiments.
  • Metal ions are necessary for the activity of many enzymes (e.g., DNases and DNA modifying enzymes) as well as interactions of biomolecules (e.g., receptor-ligand interaction). Sequestration of metal ions disrupts metal ion-dependent interactions (e.g., cell-cell and cell substratum interaction) as well as inhibits the action of metal ion dependent enzymes.
  • Here we describe steps to prepare 1 mM EDTA solution from stock EDTA solution.

Requirement

  • Reagents
    • 0.5 M EDTA solution, pH 8.0 at 25°C
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder

Composition:

1 mM  EDTA, pH 8.0 at 25°C

Objective:

Preparation of 100 ml of 1 mM EDTA solution (pH 8.0) from 0.5 M EDTA solution (pH 8.0)

Preparation:

Step 1: Calculate amount of 0.5M EDTA required for the preparation of 100 ml of 1 mM EDTA.

You need to add 0.2 ml of 0.5 M EDTA in 99.8 ml water to achieve 100 ml of 1 mm EDTA solution. (See calculation)

Step 2: Take ≈50 ml water in a measuring cylinder and add 0.2 ml of 0.5M EDTA in it.

Step 3: Adjust the final volume 100 ml with deionized/milli Q water. Mix it.

Tips: To mix solution, you can cover the measuring cylinder with parafilm and mix the solution by inverting the measuring cylinder 3 – 4 times. Take care no solution leaks out during this process. Alternatively, you can transfer solution to storage bottle and mix solution by swirling the bottle or using magnetic stirrer.

Storage:

Transfer solution to a storage bottle. You can store the solution at room temperature. Solution is stable at room temperature for long time.

 

Follow the table to prepare EDTA solution of specific concentration and volume from 0.5 M EDTA solution
Conc. / Volume 100 ml 250 ml 500 ml 1000 ml
1 mM 0.2 ml 0.5 ml 1.0 ml 2.0 ml
5 mM 1.0 ml 2.5 ml 5.0 ml 10.0 ml
10 mM 2.0 ml 5.0 ml 10.0 ml 20.0 ml
25 mM 5.0 ml 12.5 ml 25.0 ml 50.0 ml

 

Preparation of Tris saturated Phenol

Overview:

  • Phenol is a colorless crystalline solid. Upon exposure to air and light, phenol gradually turns pink to brownish color due to oxidation.
  • Oxidation products (e.g., quinones) of phenol can cause DNA damage (breakdown of phosphodiester bonds, cross-linking of nucleic acids), therefore, should be removed from the phenol by redistillation.
  • Redistillation of phenol at 182°C under nitrogen removes oxidized products from the phenol. Redistilled phenol should be frozen and kept in the dark bottle.
  • In order to utilize phenol for extraction of the nucleic acids (e.g., DNA and RNA), phenol is first equilibrated with buffer (Tris. Cl, pH 8.0) or water.
  • Tris-saturated phenol which has pH ≈8.0 is utilized for the isolation and purification of DNA.
  • To prepare Tris-saturated phenol, phenol is equilibrated with Tris.Cl (pH 8.0) solution until the pH of phenol reaches ≈8.0.
  • Often a small amount of 8-hydroxyquinoline (0.1%) is added in the phenol. 
  • 8-hydroxyquinoline is an antioxidant, which prevents oxidation of phenol. Its yellowish color also helps to identify phenolic phase from aqueous phase during the extraction process.
  • When Tris-saturated phenol or Phenol:chloroform (1:1) solution is used to extract biological samples or DNA solution, both DNA and RNA partitions into the aqueous phase, leaving most of impurities like protein and lipids in phenolic (organic) phase or at the interface.
  • If required, RNA contamination from the extracted DNA can be removed by RNase A digestion.

Requirements

  • Reagents
    • Redistilled Phenol, molecular biology grade: Stored in aliquots at -20℃.
    • 8-hydroxyquinoline
    • 0.5 M Tris.Cl buffer (pH 8.0)
    • 0.1 M Tris.Cl buffer (pH 8.0)
  • Equipment and disposables
    • Fume Hood
    • Magnetic stirrer and Magnetic stir bar
    • Glass Bottle/Beaker
    • Measuring cylinder

Objective

Preparation of Tris.Cl (pH 8.0) saturated Phenol

Precautions:
  • Phenol is volatile and caustic. Care must always be taken when handling phenol (wear lab coat, gloves and eye protection). Do all operations in fume hood.
  • Discard the waste according to your institution’s waste-disposal guidelines.
  • Avoid exposure of phenol to light. Cover the bottle/beaker containing phenol with aluminium foil.
Prior to start:

Set the water bath at 50°C in a fume hood.

Step 1: Thaw the frozen phenol by placing the bottle in 50°C water bath.

Step 2: Transfer 100 ml phenol to a beaker / bottle. (RG)

Step 3: Add ≈0.1 gram 8-hydroxyquinoline [final conc ≈0.1% (w/v)]. Mix to dissolve it.

Step 4. Add 100 ml of 0.5 M Tris.Cl (pH 8.0) to the phenol. Stir the mixture for 15 – 30 min on a magnetic stirrer at room temperature. Place the bottle/beaker in 50°C water bath and allow phase separation. Discard the aqueous phase. Repeat this step (3 – 4 times) until the pH of the aqueous phase is >7.8.

Step 5: Add 100 ml of 0.1 M Tris.Cl (pH 8.0) to the phenol.  Stir the mixture for 15 – 30 min as described in step 4. Allow phase separation and discard aqueous phase.

Step 6: Now transfer the tris saturated phenol to a glass bottle (100 ml) and add ≈20 ml 0.1M Tris.Cl over the phenol. Tris.Cl will form a thin upper layer (0.5 -1 cm).

Storage:

The Tris.Cl saturated phenol can be stored in dark at 4°C for 3 – 6 month. Periodically check the pH of phenol during storage. Discard it if the pH of the phenol is <7.5.

 

Preparation of Neutralization solution (solution III) for the isolation of plasmid by alkaline lysis method

Overview

  • Neutralization solution (solution III) is used for the isolation of plasmid DNA by alkaline lysis method.
  • Neutralization solution is nothing but a potassium acetate solution which has pH 4.8.
  • Addition of neutralization solution in lysed bacterial cells brings the pH back, resulting in precipitation of protein and genomic DNA.
  • Both plasmid and genomic DNA renatures upon addition of neutralization buffer. While plasmid DNA renatures in correct conformation due to its circular and covalent nature, therefore, remains in the solution, genomic DNA precipitates due to random association of both the strands.
  • Sodium dodecyl sulfate (SDS) of the lysis buffer reacts with Potassium acetate and form insoluble Potassium dodecyl sulfate (KDS).

Requirements

  • Reagents
    • 5 M Potassium acetate (CH3CO2K) solution
    • Glacial acetic acid
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 3 M Potassium
  • 5 M Acetate

Objective:

Preparation of 100 ml of Neutralization solution (solution III)

Preparation:

Step 1: To prepare, 100 ml of Neutralization solution, take 28.5 ml of Deionized / Milli-Q water in a 100 ml measuring cylinder.

Step 2: Add 60 ml of 5 M Potassium acetate and 11.5 ml of glacial acetic acid. Mix the solution.

Storage
  • Solution can be stored at room temperature in a tightly closed bottle for 1 year.
Applications
  • Plasmid isolation by alkaline lysis method
Follow the table To prepare Neutralization solution of various volume (10 ml, 25 ml, 50 ml and 1,00 ml).
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
5 M Potassium acetate 6.0 ml 15 ml 30 ml 60 ml
Glacial acetate acid 1.15 ml 2.875 ml 5.75 ml 11.5 ml
Water 2.85 ml 7.13 ml 14.25 ml 28.5 ml

Preparation of Lysis solution (solution II) for the isolation of plasmid by alkaline lysis method

Overview

  • Lysis solution (solution II) is used for the isolation of plasmid DNA by alkaline lysis method.
  • The plasmid-containing bacterial cells are lysed by treatment with the lysis solution.
  • Lysis solution contains sodium hydroxide (NaOH) and sodium dodecyl sulfate (SDS).
  • SDS is a detergent which solubilizes the phospholipid and denatures the protein, leading to lysis and release of the cell contents. Denaturing action of SDS also releases protein from DNA, leaving the DNA (both genomic and plasmid DNA) free from proteins.
  • High alkaline condition due to NaOH denatures the plasmid and genomic DNA.

Requirements

  • Reagents and solutions

    • 10 N Sodium hydroxide (NaOH) solution
    • 10% sodium dodecyl sulfate (SDS)
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)
Composition
  • 0.2 N Sodium hydroxide (NaOH)
  • 1% (wt/vol) Sodium Dodecyl Sulfate (SDS)

Objective

  • Preparation of 10 ml of lysis solution (solution II)

Preparation

Step 1: To prepare, 10 ml of lysis solution, take 8 ml of Deionized / Milli-Q water in a 10 ml measuring cylinder.

Step 2: Add 0.2 ml of 10 N NaOH solution and 1.0 ml of 10% sodium dodecyl sulfate (SDS).

Tip:
  • You may see some white precipitate when you add SDS. Dissolve it by mixing.
Precaution:
  • Do not mix concentrated stock solutions together. This will cause precipitation.

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

Storage
  • Solution can be stored at room temperature for a week. It is recommended to prepare fresh lysis solution for optimal lysis.
Applications
  • Preparation of plasmid DNA by alkaline lysis method
Follow the table to prepare lysis solution of various volume (10 ml, 25 ml, 50 ml and 1,00 ml).
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
10 N Sodium hydroxide (NaOH) 0.2 ml 0.5 ml 1 ml 2 ml
10% sodium dodecyl sulfate (SDS) 1.00 ml 2.5 ml 5 ml 10 ml
Water 8.8 ml 22 ml 44 ml 88 ml

Preparation of resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer containing Tris and EDTA is very common.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • EDTA  chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Resuspension buffer can be supplemented with RNase A which helps to get rid of RNA contamination from the plasmid preparation.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 95.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 95.5 ml

Preparation of RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method. It provides an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 94.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 9.45 ml 23.625 ml 47.25 ml 94.5 ml

Preparation of glucose and RNase A containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.
  • Resuspension buffer can be supplemented with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergents and chelating agents (EDTA, CDTA).
  • RNase A digest RNAs which are released from bacteria during lysis step, thus allow plasmid preparation free from RNA contamination. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases.

To know more, please the read article: Preparation of resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • 10 mg/ml RNase A
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)
  • 100 μg/ml RNase A

Objective

Preparation of 100 ml of resuspension buffer (solution I)

Preparation

Step 1: To prepare 100 ml of resuspension buffer, take 89.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0), 2.0 ml of EDTA (pH 8.0), and 1 ml RNase A. Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 3 – 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application

Preparation of plasmid DNA by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
10 mg/ml RNase A 0.1 ml 0.25 ml 0.5 ml 1.0 ml
Water 8.95 ml 22.375 ml 44.75 ml 89.5 ml

Preparation of glucose containing resuspension buffer for the isolation of plasmid by alkaline lysis method

Overview:

  • Glucose-containing resuspension buffer is used to resuspend bacterial cells during plasmid isolation by alkaline lysis method.
  • Glucose is added to make the resuspension buffer isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration.
  • Glucose-containing resuspension buffers are prone to microbial growth, therefore cannot be stored for a long time, and need to be kept at 4°C.

To know more, please read the article: Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method.

Requirements

  • Reagents
    • 1M Glucose solution, filter sterilized
    • 1M Tris.Cl (pH 8.0) solution, autoclaved
    • 0.5 EDTA (pH 8.0) solution, autoclaved
    • Deionized / Milli-Q water
  • Equipment and disposables
    • Measuring cylinder
    • Conical flask / Beaker
    • Magnetic stirrer (optional)

Composition

  • 50 mM Glucose
  • 25 mM Tris.Cl (pH 8.0)
  • 10 mM EDTA (pH 8.0)

Objective:

Preparation of 100 ml of resuspension buffer (solution I) containing glucose

Preparation:

Step 1: To prepare 100 ml of resuspension buffer, take 90.5 ml of deionized / Milli-Q water in a 100 ml measuring cylinder/beaker.
Precaution:
  • Do not mix concentrated stock solutions together. This can cause precipitation.
Step 2: Add 5 ml of 1 M glucose solution, 2.5 ml of Tris.Cl (pH 8.0) and 2.0 ml of EDTA (pH 8.0). Mix and transfer to a transparent bottle.
Tip:
  • A transparent bottle can easily be examined for any microbial growth in resuspension buffer.

Storage

The solution can be stored at 4°C for 6 months.

Precaution:
  • Frequently check the presence of any microbial growth in resuspension buffer. Discard if you detect any microbial growth.

Application:

Plasmid isolation from E.coli by alkaline lysis method

Follow the table to prepare resuspension buffer of various volume.
Reagents / Volume 10 ml 25 ml 50 ml 100 ml
1M Glucose solution 0.5 ml 1.25 ml 2.5 ml 5 ml
1M Tris.Cl (pH 8.0) 0.25 ml 0.625 ml 1.25 ml 2.5 ml
0.5 M EDTA (pH 8.0) 0.2 ml 0.5 ml 1.0 ml 2.0 ml
Water 9.05 ml 22.625 ml 45.25 ml 90.5 ml

Resuspension buffer (solution I) for isolation of plasmid by alkaline lysis method

  • Resuspension buffer (solution I) is used for the isolation of plasmid DNA by alkaline lysis method. Bacterial cells, obtained from the culture (liquid culture or colonies, grown on a agar plate), is resuspended in this buffer. The purpose of resuspension buffer is to provide an optimal starting pH (pH 8.0) and an ideal condition for subsequent lysis.
  • The classical composition of resuspension buffer (designed by Birnboim and Doly) contained Lysozyme, Glucose, Tris.Cl, and CDTA (or EDTA). Most of the recent formulations do not contain lysozyme and glucose.
  • Lysozymes are glycoside hydrolases that destroy bacterial cell walls by catalyzing the hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan. However, for most bacteria including E. coli DH5α, lysis solution was found to induce complete lysis, thus eliminated the use of lysozymes.
  • Glucose is added to make the solution isotonic. However, isotonicity is not required for cell wall containing bacteria including E. coli DH5α. Cell wall containing bacteria can withstand a wide range of solution concentration. Therefore, Glucose is not included in the recent recipes of resuspension buffer. Furthermore, glucose-containing resuspension buffer cannot be stored for a long time, and need to be kept at 4°C.
  • EDTA (or CDTA) chelates the divalent cations which are released upon bacterial lysis. Divalent cations are required for many enzymatic reactions. EDTA action results in inactivation of many enzymes which may harm plasmid DNA.
  • Tris.Cl acts as a buffering agent and maintains the pH of the resuspension buffer 8.0.
  • Now researchers prefer to supplement resuspension buffer with RNase A. RNase A is a very stable enzyme and is active under the very stringent condition including high alkaline condition, the presence of detergent and chelating agent (EDTA). Addition of RNase A in resuspension buffer helps to remove RNA from the plasmid preparation. In the subsequent lysis step, RNase A digests the RNA of the bacteria. However, such plasmid preparation cannot be used for in-vitro transcription due to contamination of RNases. In addition, RNase A containing resuspension buffer should be stored at 4°C and has a limited life (3 – 6 months).
  • pH indicator, LyseBlue from Qiagen, can also be added to the resuspension buffer. LyseBlue ensures the complete lysis and subsequently neutralization step. Both steps are very important to get high quality of plasmid DNA.
  • Resuspension buffer is not included in the protocol of plasmid isolation using plasmid isolation kit provided by some manufacturers (see Zyppy Plasmid Miniprep Kit). In those procedures, highly concentrated lysis buffer is added directly to the overnight grown liquid culture of bacterial cells.
  • The following types of resuspension buffer can be used for plasmid isolation

Preparation of freezing medium containing DMSO and FBS

Overview:

  • Freezing medium can be used to preserve cell lines for long term at ultra-low temperature. This method of preserving cell line is called cryopreservation.
  • An ideal freezing medium must allow 100% recovery without causing any change in cell characteristics after revival.
  • Freezing medium is nothing but a complete medium supplemented with high concentration of serum and a cryoprotective agent such as DMSO or glycerol.
  • As the name suggest, cryoprotective agents protect cells from lysis due to ice crystal formation at temperature below freezing point.
  • Serum concentration as high as 90% can be used in freezing medium. High serum concentration improves cell viability and recovery after thawing.
  • Serum-free chemically-defined freezing media are also available. They are very useful to preserve those cell lines which are maintained in serum-free chemically defined medium.
  • Both serum-containing and serum-free freezing medium are available commercially.
  • Serum-containing medium is used for cell lines which are maintained in serum-supplemented growth medium. We recommend to check cell line manual for optimal freezing medium.

Requirements

  • Reagents
    • Fetal bovine serum (FBS) or Fetal calf serum (FCS)
    • DMSO (sterile)
    • Complete growth medium (optional)
  • Equipment and disposables
    • 50 ml sterile polypropylene tubes
    • Pipetboy
    • Pipets
    • Laminar flow hood
Note:
  • Use the same growth medium which is used to maintain cell line (e.g., use DMEM, if the cell line is maintained in DMEM medium).

Composition:

  • 50% FBS
  • 40% complete growth medium
  • 10% DMSO

Objective:

Preparation of 50 ml serum-containing freezing medium

Note: All operations must be done under sterile condition. Wipe carefully surface of reagent containing bottles (e.g., DMSO bottle, FBS bottle) with 70% ethanol before placing them inside flow hood.

Preparation:

Step 1: To prepare 50 ml freezing medium, transfer 25 ml FBS and 20 ml growth medium to a 50-ml sterile polypropylene tube. Tighten the cap of the tube and mix by gentle inversion. Store it on ice.

Tips: You can also use sterile containers like beaker. We recommend you to use 50 ml sterile polypropylene tube.

Step 2: Step 2: Add 5 ml DMSO slowly while shaking the tube. Tighten the cap of the tube and mix by inverting the tube many times (5 – 6 times).
Note: Mixing of DMSO to serum is an exothermic reaction which can cause denaturation of serum proteins, resulting in precipitation. Therefore, it is recommended to use chilled FBS.

Precautions: Do not store DMSO on ice. It will solidify on ice.

Step 3 (optional): Check the sterility of freezing by keeping a small aliquot in a petri dish in the CO2 incubator.
Tips: We recommend you to check sterility of freezing medium.

Storage:

Store at 4°C for few days. Freezing medium is stable for at least 6 months at -20°C.

Tips: Freezing and thawing multiple times can cause proteins denaturation and precipitation.

Applications:

  • Serum-containing freezing medium is used for cryopreservation of cell lines.

Follow the table to prepare freezing medium of different volume.

Reagent/volume 10 ml 50 ml 100 ml 250 ml
FBS 5 25 50 125
DMSO 1 5 10 25
Growth medium 4 20 40 100