Monthly Archives: January 2018

Phenol-chloroform extraction of nucleic acid

  • Phenol-chloroform extraction can be used to isolate and purify DNA and RNA. It is extremely good at removing protein and lipid from the nucleic acid.
  • It is an example of liquid – liquid extraction which is based on differential solubilities of biomolecules (e.g., nucleic acids, proteins, carbohydrate, and lipids) in water and phenol/chloroform.
  • Since most proteins and lipids are highly soluble in phenol, they move to organic phase, whereas nucleic acid stay in aqueous phase due to its high solubility in water.
  • Phenol has slightly higher density (1.07 g/cm3) than water, but when chloroform is added which has comparatively very high density (1.47g/cm3), the density of the phenol-chloroform solution increases considerably. Therefore during extraction, organic phase is always present at the bottom, allowing aqueous phase to be transferred efficiently.
  • Solution of phenol and chloroform is not only more efficient at denaturing proteins but also reduces the partitioning of poly(A)+ mRNA into the the organic phase.
  • Generally a 1:1 ratio of phenol:chloroform is used for the extraction of nucleic acid. Sometime a small quantity of isoamyl alcohol is added in this mixture (25:24:1 ratio of Phenol: Chloroform: Isoamyl Alcohol). Isoamyl alcohol is a anti-foaming agent, thus prevents form-formation during the extraction process.
  • In this procedure, approximately equal volume of phenol:chloroform solution is added to  the sample (aqueous solution DNA/RNA or cell lysate or tissue lysate) and mixed. When this mixture is centrifuged, solution is separated into two phases: upper aqueous phase and bottom organic phase (Phenol/Chloroform). At interface, insoluble material which is nothing but the cell debris and denatured proteins are collected. Aqueous phase which contains nucleic acid is then transferred to another fresh vial and subjected to ethanol or isopropanol precipitation to concentrate nucleic acid.
  • Tris saturated phenol (pH ≈8.0) is used for purification of DNA, whereas water saturated phenol (pH ≈4.8) is used for purification of RNA.
  • At slightly alkaline pH, phosphate backbone of both DNA and RNA is negatively charged, therefore, they remains in aqueous phase. As the pH of Phenol/Chloroform solution drops, DNA tend to move to organic phase and at pH 4.8 most DNA is present either at interphase (mostly large DNA fragments) or in the organic phase (smaller DNA fragments). The reason why DNA move to organic phase, but not RNA is because of higher pKa value of DNA, which results in neutralization of negatively charged phosphate backbone of DNA.
  • Since phenol is slightly soluble in water (≈7-8% in water), aqueous phase can be reextracted with chloroform alone to remove traces of phenol from the final preparation of nucleic acid.  Traces of phenol can inhibit downstream enzymatic reactions and interfere with spectroscopic analysis.

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.

 

Protocol – Plasmid isolation by boiling method (miniprep)

Overview

  • The boiling lysis method of plasmid isolation is quick and is recommended for isolation of small plasmids (up to 10 kb). Plasmids larger than 10 kb should be isolated by other methods (e.g., alkaline lysis method)
  • The quality of plasmid, isolated by this method, is not as good as the plasmid isolated by alkaline lysis method. However, the quality is good enough for restriction digestion analysis. It’s rapidity together with the plasmid quality, suitable for restriction digestion analysis, makes this a method of choice for screening of large number of clones during cloning experiments.
  • This method is not suitable for isolating plasmids from E. coli endA + strains (e.g., HB101, JM100).
  • In this method, the bacterial cells are given brief heat treatment in boiling water bath in presence of lysozyme and triton X-100. Plasmid DNA, due to its small size, comes out from the bacterial cell, whereas, genomic DNA remains trapped inside the cell.
  • Subsequent high speed centrifugation separates the plasmid DNA from rest of the cell debris, which form pellet. Pellet is removed and plasmid DNA is recovered by ethanol or isopropanol precipitation method.

Requirements

  • Reagents and solutions
    • STE solution [8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0)]
    • STET solution [8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 5% (w/v) Triton X-100)]
    • Lysozyme stock solution [10 mg/mL Lysozyme in 10 mM Tris-HCl (pH 8.0)]
    • Phenol : Chloroform : Isoamyl alcohol (25 : 24 : 1) solution (optional)
    • 70% Ethanol
    • Isopropanol
    • Tris – EDTA (TE) (100 mM Tris, 10 mM EDTA, pH 8.0)
    • DNase free RNase A (10 mg/ml)
  • Equipment and disposables
    • Boiling water bath
    • Microcentrifuge tubes
    • Micropipette and tips
    • Ice
    • Gloves

Objective

  • Isolation of plasmid DNA from 1-3 ml of bacterial culture (E. coli DH5α) by boiling lysis method.
Starting material:
  • 3 ml overnight grown culture of E. coli DH5α containing plasmid of interest.
Prior to start:
  • Make sure that STE and STET solutions are chilled
  • Set the centrifuge for cooling (4°C)

Protocol:

Step 1: Harvest bacterial cells from 1.5 ml culture
  • Pour 1.5 ml overnight grown culture in a microcentrifuge tube.
  • Centrifuge at room temperature (or 4°C) for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min).
  • Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.
Notes:
  • The yield of plasmid DNA is dependent mainly on the copy number of the plasmid. For high copy number plasmid, 1.5 ml culture is sufficient to get a good yield of plasmid DNA. However, more culture is required for good yield of low-copy-number plasmid.
Tips:
  • To remove the medium completely, decant the medium from the microcentrifuge tube after centrifugation. Invert microcentrifuge tube upside down on a paper towel to remove residual liquid. Tap the tube gently on the paper towel to remove liquid sticking on the sides of the tube.
  • To take more bacterial culture (more than 1.5 ml) for plasmid isolation, repeat the above process by adding more culture in the same microcentrifuge tube. Microcentrifuge tube with 2 ml capacity can also be used.
  • Chloramphenicol treatment can be used to amplify low-copy number plasmid.
Precautions:
  • While harvesting the bacteria, the speed of centrifugation and time should be optimized in such a way that the pellet after centrifugation should be loose and at the same time supernatant should be clear. If the pellet is tight, it would be difficult to make the suspension of the pellet. Generally, above mention condition works well.
  • Try to remove medium from the pellet completely. Traces of medium may inhibit some of the sensitive restriction enzymes action.
Step 2 (Optional): Wash the bacterial cells with STE solution
  • Add 500 μl ice cold STE solution.
  • Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.
  • Centrifuge at 4°C for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min).
  • Remove the supernatant from the tube completely.
Notes:
  • The purpose of this step is to remove traces of culture medium from the bacterial cells, which otherwise can cause inhibition of some sensitive restriction enzyme reaction.
Tips:
  • Tris – EDTA solution (100 mM Tris, 10 mM EDTA, pH 8.0) can also be used in place of STE solution to wash the pellet.
Step 3: Resuspend bacterial pellet in STET solution
  • Add 350 μl of STET solution and resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.
Precautions:
  • Make sure that the bacterial pellet is completely dispersed in STET solution. No cell clumps should be visible before the boiling in the water bath. Clumps can cause low yield of plasmid.
Step 4: Treat bacterial cells with lysozyme.
  • Add 25 μl of freshly prepared solution of lysozyme and mix immediately by vortexing for 5 seconds.
Precautions:
  • Lysozyme will not work efficiently if the solution pH is less than 8.0.

Step 5: Now place the tube in a boiling water bath for approximately 1 min (40 sec – 60 sec).

Step 6: Clear the lysate by high-speed centrifugation
  • Centrifuge the tube at maximum speed (14,000 rpm) in a microcentrifuge for 10 min at 4°C or room temperature.
  • Transfer the supernatant containing plasmid promptly in new microcentrifuge tube. Alternatively, one can remove the viscous pellet with a sterile toothpick.
Notes:
  • One can centrifuge the tube at room temperature. Centrifugation at 4°C generates tight pellet than centrifugation at room temperature. The tight pellet can be removed easily.
Precautions:
  • While transferring the supernatant or removing the pellet with a sterile toothpick, take care that debris should not come along with the supernatant. Supernatant should be centrifuged again if it contains any suspended particle.
Step 7 (Optional): Extract the supernatant with Phenol : Chloroform : isoamylalcohol solution.
  • This step will remove impurities including protein and lipid contamination from the plasmid preparation.
  • Add equal volume of Phenol:Chloroform:Isoamylalcohol (25:24:1) in the supernatant. Mix by vortexing for 10 sec. Centrifuge at maximum speed at 4°C. Transfer the supernatant to fresh microcentrifuge tube.
Precautions:
  • While transferring the supernatant, take care that no traces of phenol come along with supernatant. Traces of phenol is sufficient to inhibit most enzymatic reactions.
  • Phenol and chloroform are toxic. Follow the safety rules while handling phenol.
Step 8: Recover plasmid from supernatant by isopropanol precipitation.
  • Add equal volume of isopropanol in the supernatant.
  • Mix it by inverting the tube 4 – 6 times. C
  • entrifuge at maximum speed (14,000 rpm) for 30 min at 25°C.
  • Remove the supernatant completely.
Precautions:
  • Incubation of the above mix at room temperature or on ice increases plasmid yield but also causes salt precipitation. Above mentioned condition generally gives good quality of plasmid DNA without much salt contamination.
  • While removing the supernatant, care should be taken as isopropanol precipitated plasmid pellet is loosely attached to the surface and is invisible in most cases. Careless removal of supernatant can result in loss of plasmid pellet.
Step 9: Wash the pellet with 70% ethanol.
  • Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times.
  • Centrifuge at 14,000 rpm (maximum speed) for 10 min at 25°C.
  • Remove the supernatant completely.
Tips:
  • To remove the supernatant, one can decant the supernatant after first centrifugation. Remains of liquid will be sticking on the wall of microcentrifuge tube. A second flash spin is sufficient to collect all the liquid at the bottom which can be removed by pipetting. Air dry the pellet for 5 min.
Precautions:
  • Take care with this step, as the pellet sometimes does not adhere tightly to the tube and lost while removing the supernatant.
  • Do not overdry the pellet. Overdried pellet is difficult to dissolve.
  • Remove the traces of ethanol as it may inhibit some enzyme reactions.

Step 10: Dissolve the pellet in 25 μl sterile double distilled water or TE (pH 8.0).

Tips:
  • To dissolve the pellet, one can vortex the solution gently for a brief period and also can incubate at 37° for ∼20 minutes.

Storage

  • Solution can be stored at 4°C for few days. Store at -20°C for years.
Precautions:
  • Don’t thaw the plasmid repeatedly. This can cause reduction of the supercoiled form of the plasmid.

Applications

  • The isolated plasmid is suitable for most of our cloning experiments. Often the amount of supercoiled plasmid is comparatively less, therefore, is not suitable for transfection experiments.

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

Plasmid isolation

  • Plasmid isolation is a routine task in most cell and molecular biology labs. It is an essential step in many procedures such as gene cloning, DNA sequencing, and transfection.
  • A good method of plasmid isolation must be rapid, economical and produce plasmid of high quality which can be used for multiple purposes including transfection in cell lines, sequencing, and cloning.
  • To isolate plasmid from the host bacteria, cells are first lysed, leading to release of plasmid and in subsequent steps plasmid is purified from the lysate.
  • There are number of methods available to lyse bacterial cells. Most common methods are alkaline lysis, boiling lysis, enzymatic lysis and lysis with detergents.
  • Purification of plasmid from the lysed cells mostly dependent on the type of lysis method used to release plasmid in solution. For example, alkaline lysis which completely disrupt the bacterial cells leading to release of cell components including both plasmid DNA and genomic DNA in denatured state, rely on selective renaturation of only plasmid DNA in a perfect manner at purification step. On the other hand, boiling lysis selectively releases only plasmid DNA from the bacterial cells.
  • Purified plasmid can be further purified by number of methods to obtain high quality of plasmids. These methods are selective precipitation in high salt SDS, centrifugation in gradients of CsCl – ethidium bromide (EtBr), extraction with Phenol-chloroform, and hydroxylapatite chromatography.
  • Following factors can be considered while choosing a method of plasmid isolation isolation……
    • Host strain
    • Plasmid characteristics (copy number and size)
    • Quality and quantity of plasmid
    • Complexity, cost and rapidity of the method
  • The yield of plasmid DNA is governed by two most important parameters – copy number of plasmid and amount of initial culture taken to isolate plasmid DNA. Based on initial culture volume, plasmid isolation method can be termed as…….
    • Miniprep (1 – 5 ml culture volume)
    • Midiprep (25 – 50 ml culture volume)
    • Maxiprep (100 – 500 ml culture volume)
    • Megaprep (1000 – 2500 ml culture volume)
    • Gigaprep (5000 – 10000 ml culture volume)

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