13.6: Lab Technique - Bacterial Transformation

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    Transforming Bacteria with Plasmid DNA

    Bacterial transformation is a foundational technique in molecular cloning for the introduction of DNA into bacterial cells. Bacterial transformation involves two basic steps

    1. Preparation of Competent Bacteria
    2. Transformation of Competent Bacteria: can be performed either through chemical transformation or through electrical transformation (i.e. electroporation)

    Step #1: Preparation of Competent Bacteria

    The most common bacteria used for transformation is the DH5α strain of E.coli. However, other E.coli strains are also used. Because most bacterial strains used for bacterial transformation have a low level of "natural competency", they must be made competent. Competent bacterial cells are bacteria that have been "weakened" or made competent to allow for the entrance of the DNA. The preparation of competent cells for chemical transformation (i.e., chemically competent cells) is done using ice-cold salt solutions, whereas competent cells for electroporation (i.e., electrically competent cells) are prepared with ice-cold water. Both procedures require two days to complete. On the first day, a starter culture is grown overnight in a suitable liquid medium that does not contain any selective agents (e.g. antibiotics). On day 2, this starter culture is used to inoculate a larger volume of the same liquid medium. This subculture is grown until it reaches an optical density (OD) at 600 nm (i.e., OD600) of 0.4 to 0.9. The cells are then harvested and made competent. This optical density ensures that the bacteria are at the midpoint of their logarithmic growth phase. It is crucial that the bacterial cells are in logarithmic phase before they are made competent. The protocol given below uses ice-cold, sterile solutions. Once the cells are exposed to an ice-cold solution, they are in a weakened state and should be treated with care. Re-suspensions should be done slowly and gently and centrifugation should be done at a low enough speed to not damage the cells, but allow for pelleting.

    Lab Protocol: Preparation of Competent Cells

    1. Inoculate 2.0 mL of liquid medium (e.g. LB) with a single fresh bacterial colony, using sterile technique, and grow overnight at 37°C with shaking at 200 to 250 rpm. Do not include antibiotics in this liquid culture.
    2. Using sterile technique, subculture the 2.0 mL starter culture by adding it to 1.0 L of the same liquid medium. Grow at 37°C with shaking at 200 to 250 rpm. At regular intervals, remove a small aliquot of this subculture using sterile technique and measure its absorbance in a spectrophotometer at 600 nm. Discard the aliquot. Culture until the medium reaches an optical density of 0.4 to 0.9.
    3. Centrifuge the cells for 10 minutes at 5,000 rpm at 4°C to pellet.
    4. Remove the supernatant completely.
    5. Place the cells on ice.
    6. To make chemically-competent cells:
      1. Re-suspend each pellet in 5.0 mL of sterile, ice-cold 10 mM CaCl2 and combine the two re-suspensions into one tube. Place on ice.
      2. Centrifuge the cells for 5 minutes at 2,500 rpm at 4°C to pellet.
      3. Remove the supernatant completely.
      4. Gently re-suspend the pellet in 5.0 mL of sterile, ice-cold 10 mM CaCl2 and incubate on ice for 30 minutes.
      5. Centrifuge the cells for 5 minutes at 2,500 rpm at 4°C to pellet.
      6. Remove the supernatant completely.
      7. Gently re-suspend the pellet in 2.0 mL of sterile, ice-cold 10 mM CaCl2.
    7. To make electrically-competent cells
      1. Re-suspend each pellet in 5.0 mL of sterile, ice-cold distilled water and combine the two re-suspensions into one tube. Place on ice.
      2. Centrifuge the cells for 5 minutes at 2,500 rpm at 4°C to pellet.
      3. Remove the supernatant completely.
      4. Gently re-suspend the pellet in 5.0 mL of sterile, ice-cold distilled water and incubate on ice for 30 minutes.
      5. Centrifuge the cells for 5 minutes at 2,500 rpm at 4°C to pellet.
      6. Remove the supernatant completely.
      7. Gently re-suspend the pellet in 2.0 mL of sterile, ice-cold distilled water. Place cells on ice.
    8. Aliquot the cells into 50 µL aliquots.
    9. Store aliquots at -80°C.
    Competency Reagents

    Transformation Medium (10 mM CaCl2)

    • dissolve 1.47 g of CaCl2 in 1000 mL of water
    • filter through a 10 µm filter to sterilize
    • store at 4°C

    Sterile Water

    • autoclave 1.0 L of distilled water to sterilize
    • store at 4°C

    Step #2: Transformation of Bacteria

    Transformation can be done chemically, through the use of ice-cold salt solutions and a rapid "heat shock", or electrically, using an electroporator. Once the bacteria have been transformed, the bacterial samples are plated onto the desired selective media for colony growth. How well the bacterial cells take up DNA can be measured by calculating transformation efficiency. Transformation efficiency is defined as the number of colony forming units (cfu) produced by transforming 1 µg of plasmid into a given volume of competent cells. It is calculated using the formula:

    • Transformation efficiency (# transformants per µg DNA) = (# of colonies/µg DNA) x (mL of recovery volume used/mL of volume plated)

    Chemical transformation relies upon a "heat shock" of ice-cold bacteria to weaken the cell membrane, permitting DNA entry. This heat shock is performed for a short period of time at 42°C. Electrical transformation through electroporation introduces DNA into bacterial cells by means of a short pulse of electricity that decreases exponentially over the course of the electroporation. This short pulse introduces tiny pores in the cell membrane, allowing the entrance of DNA into the cell. Because of this electric field, it is vital that all salt contamination from the competent cells and DNA preparations are absent. Excess salt in the competent cell aliquot will cause "arcing" upon electroporation, killing the cells. DNA ligations with plasmid DNA are not likely to have significant salt contamination. However, purified plasmid DNA samples should be re-suspended in nuclease-free water following their purification if electroporation is desired at some point. Electroporation requires the use of electroporation cuvettes and an electroporator (Figure \(\PageIndex{1}\)).

    picture of an electroporator and image of the electroporation cuvette
    Figure \(\PageIndex{1}\): The electroporator electrically transforms cells. (A) The electroporator is a piece of lab equipment that delivers an electrical pulse to a cell suspension, causing the entrance of DNA into the cells. (B) The disposable electroporation cuvette contains a chamber for the cell suspension and makes electrical contact with the electroporator through embedded electrodes. (Electroporation by Patricia Zuk, CC BY 4.0)

    Following chemical transformation or electroporation, the transformed cells are allowed to "recover" in a nutrient-rich medium at 37°C for a short period of time with gentle shaking before plating onto the appropriate growth medium. SOC media, containing glucose and MgCl2, is the preferred nutrient-rich medium for this recovery as it is known to increase the transformation efficiency of transformed cells more than LB broth. However, LB broth may be used.

    The protocols given below describes the transformation of competent bacteria with plasmid ligations. However, any type of plasmid DNA preparation can be introduced into competent bacteria. The amount of DNA to be added to a competent cell aliquot should range from 1 ng to 10 ng per aliquot.

    Lab Protocol: Chemical Transformation of Bacteria

    1. Prior to transformation, remove the desired number of competent cell aliquots from the –80°C and thaw the cells slowly on ice. Pre-warm the required number of agar plates to 37°C.
    2. As a positive control for transformation, dilute a plasmid, such as pUC19, to 10 pg/μL using sterile water.
    3. Once thawed, immediately add 1.0 µL of each ligation (or plasmid DNA sample) to a 50 µL aliquot of chemically-competent cells.
    4. Place the aliquot on ice for 5 minutes.
    5. Heat shock cells at 42°C for exactly 30 seconds.
    6. Immediately place the cells back on ice for 5 minutes.
    7. Add 950 µl of room temperature SOC media (or LB Broth) to each cuvette, gently mix up and down twice to mix, then transfer to sterile round-bottom culture tubes (e.g. 17 mm x 100 mm).
    8. Place the transformed cells onto a shaking incubator platform and incubate for 1 hour at 37°C while shaking at 200 to 250 rpm.
    9. Plate 100 µL of the transformed cells onto an LB agar plate containing the appropriate antibiotic. Allow to "soak" into the agar. Label the plates accordingly.
    10. Incubate the plates overnight in an inverted position in a 37°C bacterial incubator.

    Lab Protocol: Electroporation of Bacteria

    1. Prior to electroporation, place sterile SOC media (or LB broth) in a 37°C water bath. Place the desired number of electroporation cuvettes (0.1 cm) and 1.5 mL microfuge tubes on ice. Pre-warm the required number of agar plates to 37°C.
    2. As a positive control for transformation, dilute the control plasmid pUC19 to 10 pg/μL using sterile water.
    3. Thaw the desired number of aliquots of electrically-competent cells on ice.
    4. For each ligation (or plasmid DNA sample), transfer 50 μL of competent cells into a chilled electroporation cuvette. Add 1 μL of DNA into each cuvette.
    5. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells are spread across the bottom of the cuvette.
    6. Electroporate using the following conditions: 2.1 kV, 100 Ω, and 25 μF. The typical time constant is ~2.6 milliseconds.
    7. Immediately add 950 µl of pre-warmed SOC media (or LB Broth) to each cuvette, gently mix up and down twice to mix, then transfer to sterile round-bottom culture tubes (e.g. 17 mm x 100 mm).
    8. Place the transformed cells onto a shaking incubator platform and incubate for 1 hour at 37°C while shaking at 200 to 250 rpm.
    9. Plate 100 µL of the transformed cells onto an LB agar plate containing the appropriate antibiotic. Allow to "soak" into the agar. Label the plates accordingly.
    10. Incubate the plates overnight in an inverted position in a 37°C bacterial incubator.
    Transformation Reagents

    SOC Media

    • dissolve the following in 500 mL distilled water
      • 10 g of tryptone (2% final)
      • 2.5 g of yeast extract (0.5% final)
      • 0.29 g of NaCl (10 mM final)
      • 0.095 g of KCl (2.5 mM final)
      • 1.015 g of MgCl2 (10 mM final)
      • 0.6 g of MgSO4 (10 mM final)
      • 1.8 g of glucose (20 mM final)
    • autoclave to sterilize
    • store at 4°C
    • final concentration:
      • 2% tryptone
      • 0.5% Yeast Extract
      • 10 mM NaCl
      • 2.5 mM KCl
      • 10 mM MgCl2
      • 10 mM MgSO4
      • 20 mM glucose

    LB Broth (Miller)

    • dissolve the following in 1 L distilled water:
      • 10 g of tryptone
      • 5 g of yeast extract
      • 10 g of NaCl
    • autoclave to sterilize
    • store at 4°C
    • final concentration:
      • 1% tryptone
      • 0.5% yeast extract
      • 1% NaCl

    LB amp agar plates

    • dissolve the following in 1 L distilled water:
      • 10 g of tryptone
      • 5 g of yeast extract
      • 10 g of NaCl
      • 15 g of agar
    • autoclave to sterilize
    • cool to 55°C and add antibiotic to the desired concentration (e.g. 100 mg ampicillin; 100 µg/ml final)
    • once poured, store at 4°C
    • final concentration:
      • 1% tryptone
      • 0.5% yeast extract
      • 1% NaCl
      • 1.5% agar
      • 100 µg/ml ampicillin

    13.6: Lab Technique - Bacterial Transformation is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by LibreTexts.