Chemical Transformation Tips

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  • Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 µg of plasmid into a given volume of competent cells. The term is somewhat misleading in that 1 µg of plasmid is rarely actually transformed. Instead, efficiency is routinely calculated by transforming 100 pg-1 ng of highly purified supercoiled plasmid under ideal conditions. The equation for calculating Transformation Efficiency (TE) is: TE = Colonies/µg/Dilution. Efficiency calculations can be used to compare cells or ligations. We have listed our recommended protocols and tips to help achieve maximum results.

    Recommended Protocols

    High Efficiency Transformation Protocol

    1. Thaw cells in single-use transformation tubes on ice for 10 minutes. For 200 µl tubes, thaw on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
    2. Add 1 pg-100 ng of plasmid DNA (1-5 µl) to cells and mix without vortexing.
    3. Place on ice for 30 minutes.
    4. Heat shock at 42°C for 30 seconds.*
    5. Place on ice for 5 minutes.
    6. Add 950 µl of room temperature SOC.
    7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
    8. Mix cells without vortexing and perform several 10-fold serial dilutions in SOC.
    9. Spread 50-100 µl of each dilution onto pre-warmed selection plates and incubate at 37°C or according to recommendations.

      * Exactly 10 seconds for BL21 (NEB #C2530)

    5 Minute Transformation Protocol

    Results in only 10% efficiency compared to above protocol.

    1. Thaw cells in your hand.
    2. Add 1 pg-100 ng of plasmid DNA (1-5 µl) to cells and mix without vortexing.
    3. Place on ice for 2 minutes.
    4. Heat shock at 42°C for 30 seconds.
    5. Place on ice for 2 minutes.
    6. Add 950 µl of room temperature SOC. Immediately spread 50-100 µl onto a selection plate and incubate overnight at 37-42°C. NOTE: Selection using antibiotics other than ampicillin may require some outgrowth prior to plating.

    Transformation Tips

    Thawing

    • Cells are best thawed on ice.
    • DNA should be added as soon as the last trace of ice in the tube disappears. 
    • Cells can be thawed by hand, but warming above 0°C decreases efficiency.

    Incubation of DNA with Cells on Ice

    • Incubate on ice for 30 minutes. Expect a 2-fold loss in TE for every 10 minutes you shorten this step. 

    Heat Shock

    • Both temperature and time are specific to the transformation volume and vessel. Typically, 30 seconds at 42°C is recommended, except when using BL21 (NEB #C2530 ) which requires exactly 10 seconds. 

    Outgrowth

    • Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. 
      Expect a 2-fold loss in TE for every 15 minutes you shorten this step. 
    • SOC gives 2-fold higher TE than LB medium. 
    • Incubation with shaking or rotating the tube gives 2-fold higher TE.

    Plating

    • Selection plates can be used warm or cold, wet or dry with no significant effects on TE. 
    • Warm, dry plates are easier to spread and allow for the most rapid colony formation.

    DNA

    • DNA for transformation should be purified and resuspended in water or TE Buffer. 
    • Up to 10 µl of DNA from a ligation mix can be used with only a 2-fold loss of efficiency. 
    • To maximize transformants, purification by either a spin column or phenol/chloroform extraction and ethanol precipitation should be performed. 
    • The optimal amount of DNA is lower than commonly recognized. Using clean, supercoiled pUC19, the efficiency of transformation is highest in the 100 pg-1 ng range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 ng.

    DNA Contaminants to Avoid

    Contaminant Removal Method
    Detergents Ethanol precipitate
    Phenol Extract with chloroform and ethanol precipitate
    Ethanol or Isopropanol Dry pellet before resuspending
    PEG* Column purify or phenol/chloroform extract and ethanol precipitate
    DNA binding proteins*
    (e.g., Ligase)
    Column purify or phenol/chloroform extract and ethanol precipitate

    * Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.