Monthly Archives: January 2018

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.

Growing E. coli for the plasmid isolation

  • Amplification of plasmid is desirable for many applications including gene cloning, DNA sequencing, transfection, and probe preparation. Fastest and routinely used method to amplify plasmid is to introduce plasmid in an appropriate strain of E. coli e.g. DH5α (the process is called transformation), grow them to a suitable culture volume, and finally, extract plasmid from them (the process called plasmid isolation).
  • Alternatively, E. coli DH5α harboring a plasmid can also be revived from the stored stocks e.g., glycerol stock or stab culture, if available. Cells obtained from stored stock can be either streaked or plated on an antibiotic containing solid LB-agar plate.
  • Plasmid copy number and culture volume are the two most important parameter which predicts the quantity of the plasmid extracted at the end of the isolation process. Comparatively, large culture volume is required for low copy number plasmid.
  • Plasmid copy number can be increased by chloramphenicol treatment. Several rich growth media can also be used to grow bacteria. These media support high cell density due to nutrient enrichment.
  • Depending on the initial culture volume, plasmid isolation methods are called miniprep (1-5 ml culture volume), midiprep (25-50 ml culture volume), and maxiprep (100-500 ml culture volume).
  • Generally miniprep yields sufficient amount of plasmid for applications like the screening of clones for the presence of insert, DNA sequencing etc. Other applications, like probe preparations, plasmid distribution, transfection, etc., can require a large quantity of plasmid (midiprep or maxiprep).
  • For miniprep, a single colony from the LB-agar plate is inoculated into a antibiotic-containing liquid medium. Culture is grown at 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. Incubating culture for a long time can cause the death of bacteria, which can result in low yield of plasmid. Sometimes, a well-grown colony from the LB-agar plate can directly be utilized for plasmid miniprep.
  • For a large amount of culture which is required for midiprep and maxiprep, initially, a starter culture is prepared by inoculating a small amount of culture medium (2 – 10 ml) with a single colony. When the culture reaches mid- to late-exponential growth phase (takes 8 – 12 h), culture is diluted in a ratio of 1:100 to 1:1000 to prepare large culture volume for midiprep and maxiprep.
  • All plasmid vectors carry at least one antibiotic resistance gene, which enables bacteria to survive and grow in presence of a respective antibiotic. Antibiotic functions as a selective marker which allows growth of only plasmid containing E. coli cells. In absence of antibiotic, bacteria will lose the plasmid, which will result in low or no yield.

Related Notes:

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