Category Archives: Protocols

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

Protocol – Growing large volume of E. coli culture for large scale plasmid isolation

Overview:

  • Large scale isolation of plasmid requires large volume of E. coli culture. In DNA cloning experiments, a large amount of plasmid is prepared after confirming the presence of right sequence by restriction digestion and sequencing.
  • Large scale plasmid isolation procedures are termed, midiprep (25 – 50 ml starting culture volume) and maxiprep (100 – 500 ml starting culture volume).
  • A starter culture is initially prepared by inoculating a colony in a small volume (2 – 10 ml) of culture medium.
  • Large culture volume is prepared by diluting starter culture in a ratio of 1: 100 to 1: 1000 in the growth medium.
Note:
  • Here we have taken an example of preparing liquid culture from a colony of E. coli DH5α, transformed with the pcDNA plasmid. The pcDNA plasmid carries ampicillin resistance gene, therefore, requires ampicillin for selection of plasmid-containing bacteria.
  • If your plasmid carries another antibiotic resistant gene, add the respective antibiotic in the culture medium.

Requirements

  • Reagents
    • LB medium
    • Ampicillin (Stock conc 100 mg/ml)
    • A 25-ml conical flask with cotton plug (autoclaved)/Falcon polypropylene tubes (Cat No. #352059)
    • A 500-ml conical flask with cotton plug (autoclaved)
  • Equipment and disposables
    • Bunsen burner
    • Clean workbench
    • Autoclaved toothpicks/Pipette tips/Inoculation loop

Objective:

Growing large volume of culture (100 ml) of E. coli harboring pcDNA plasmid for large scale plasmid isolation

Starting material: Grown bacterial colony on ampicillin-containing LB-Agar plate
Prior to start: Set the shaking incubator at 37°C.
  • Perform all microbiological operations close to the flame of Bunsen burner in a clean place, wiped with 70% ethanol.
  • Do all operations aseptically and use sterile material and reagents. All operation which involves opening of media bottle should be done quickly to reduce the risk of contamination. Before starting your work, clean your hands with soap.

Procedure:

A. Preparation of Starter Culture
Step 1: Prepare LB medium with antibiotic for starter culture
  • Transfer 5 ml LB medium aseptically to a 25-ml conical flask. You can use sterile pipette to transfer liquid medium into the tube.
  • Add 5 µl of ampicillin antibiotic stock solution (100mg/ml). Swirl the flask. The final concentration of kanamycin will be 100 µg/ml.
Precautions:
  • Whenever you open media bottle, show the mouth of the bottle to the flame.
Step 2: Inoculate culture medium with bacterial colony
  • Touch the surface of a bacterial colony with a sterile toothpick or pipette tip
  • Drop it into the antibiotic-containing LB medium.
Precaution:
  • Don’t inoculate culture medium directly from glycerol stock. This can cause low yield and unpredictable result.
  • Make sure that at least some bacterial cells stick to toothpick/pipette tip while picking up the colony from the LB-Agar plate.
Step 3: Grow the culture for 8 – 12 h at 37°C with vigorous shaking.
  • Set the flask in shaker incubator.
  • Set the speed 200 – 300 rpm and start the shaker. Incubate for 8 – 12 h.
Note:
  • Incubation for 8 h is sufficient to see turbidity. At this growth stage, the culture will be in log phase of the growth curve, which represents exponential growing cells. When these cells are diluted, they will maintain their exponential growth.
Tip:
  • A good way to prepare starter culture is to inoculate colony in the morning. Starter culture will be ready in the evening.

B. Preparation of large volume of culture by diluting starter culture in a ration of 1:100 to 1:1000 in growth medium

Step 5: Prepare 100 ml LB medium with antibiotics
  • Transfer 100 ml LB medium aseptically to 500-ml conical flask.
  • Add 100 µl of ampicillin stock solution. Swirl the flask. The final concentration of ampicillin will be 10 µg/ml.

Step 6: Transfer 1 ml starter culture aseptically to 100 ml LB medium.

Step 7: Grow the culture overnight (12 – 16 h) at 37°C with vigorous shaking.
  • Set the flask in the shaker incubator.
  • Set the speed 200 – 300 rpm.
  • Incubate for 12 – 16 h.

Step 8: Take out the culture next moning. Culture is ready for plasmid isolation.

Protocol – Growing liquid culture of E. coli for plasmid miniprep

Overview:

  • Small-scale plasmid isolation procedure, the miniprep, yields sufficient amount of plasmid for the screening of clones and DNA sequencing. Once a clone is confirmed for the presence of insert with right sequence, a large amount of plasmid can be prepared by midiprep or maxiprep.
  • Miniprep requires a small amount of culture of the plasmid-containing bacterial cells. Most often a single colony from the LB-agar plate is inoculated in a liquid medium. Culture is grown at the 37°C in a shaker incubator overnight (12- 16 h). Grown culture corresponds to late log phase/early stationary phase of bacterial growth and is characterized by low content of RNA. At this stage, the grown culture has a density of 3 – 4 × 109 cells/ml.
  • Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid isolation. A well-grown colony on LB-agar plate is prepared by streaking a colony in a small area (0.5 – 1 cm long). This is more convenient when you need to screen a large number of colonies.
  • An antibiotic should be present at all stages of culture growth. The choice of antibiotic depends on the antibiotic resistant gene carried by the plasmid. In absence of antibiotic, dividing cells can lose the plasmid, resulting in low plasmid yield.
Note:
  • Here we have taken an example of preparing liquid culture from the a colony of E. coli DH5α, transformed with the pEGFP plasmid. The pEGFP plasmid contains kanamycin resistance gene, therefore, requires kanamycin for selection of plasmid-containing bacteria.
  • If your plasmid carries another antibiotic resistant gene, add the respective antibiotic in the culture medium.

Requirements

  • Reagents
    • LB medium
    • Kanamycin (Stock conc. 50 mg/ml)
    • Falcon® 14mL Round Bottom polypropylene tube with Snap Cap (Cat No. #352059)/ 25-ml conical flask with cotton plug (autoclaved)
  • Equipment and disposables
    • Bunsen burner
    • Clean workbench
    • Autoclaved toothpick/Pipette tips/Inoculation loop

Objective:

Growing liquid culture of E. coli DH5α harboring pEGFP plasmid for miniprep

Starting material: Bacterial colony on antibiotic containing LB-Agar plate
Prior to start: Set the shaking incubator at 37°C.
  • Perform all microbiological operations close to the flame of bunsen burner in a clean place, wiped with 70% ethanol.
  • Do all operations aseptically and use sterile material and reagents. All operation which involves opening of media bottle should be done quickly to reduce the risk of contamination. Before starting your work, clean your hands with soap.

Procedure:

Step 1: Prepare LB medium with antibiotics
  • Transfer 3 ml LB medium aseptically to polypropylene tube (Falcon, Cat No. #352059).
  • Add 3 µl of antibiotics stock solution of kanamycin (50 mg/ml). The final concentration of kanamycin will be 50 µg/ml.
Note:
  • A single colony can be inoculated in 2 – 10 ml culture volume. Since miniprep needs 1 – 3 ml culture, inoculating 3 ml culture medium is sufficient.
  • Disposable plastic tubes with Snap Cap is a good choice for culture vessel. These tubes are available in ready to use form (sterile), easy to cap and provide good aeration and are cheap. These tubes can be discarded after use, therefore, no effort is required for cleaning and preparing them for the next use. Conical flasks and glass test tubes can also be used for culturing bacteria.
  • Depending on how many colonies you want to inoculate, prepare the same number of flasks. If you are screening for the presence of an insert in a plasmid, you need to inoculate many colonies in separate polypropylene tubes. For example, if you need to inoculate 10 colonies, you must prepare 10 polypropylene tubes with culture medium. In this case, you need 30 ml culture medium. Take 30 ml culture medium, add 30 µl kanamycin and distribute 3 ml in each polypropylene tube.
Tips: 
  • You can either pour or use sterile pipette to transfer liquid medium into the tube. Since Falcon polypropylene tubes (Cat No. #352059) have markings, pouring is more convenient and quick if you have many colonies to inoculate.
Precautions:
  • Whenever you open media bottle, show the mouth of the bottle to the flame.
Step 2: Inoculate culture medium with bacterial colony
  • Touch the surface of a bacterial colony with a sterile toothpick or pipette tip
  • Drop it into the antibiotic containing LB medium.
Precaution:
  • Don’t inoculate culture medium directly from glycerol stock. This can cause low yield and unpredictable result.
  • Make sure that at least some bacterial cells stick to toothpick/pipette tip while picking up the colony from the LB-Agar plate.
Step 3: Grow the culture overnight (12 – 16 h) at 37°C with vigorous shaking.
  • Set the culture tube in shaker incubator. Use appropriate inclined (30° – 45°) angle if you are using polypropylene tube to ensures good shaking.
  • Set the speed 200 – 300 rpm and start the shaker. Incubate for 12 – 16 h.
Precaution:
  • Don’t grow more than 16h.
Step 4: Take out the culture next moning. Culture is ready for plasmid isolation.
 
Note:
  • It is always good to start the isolation process immediately or in the same day. Culture can be stored for 2 – 3 days at 4°C. Longer storage may cause low plasmid yield.

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