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Molecular Cloning Center

Plasmid or vector transformation is the process by which exogenous DNA is transferred into the host cell. Transformation usually implies uptake of DNA into bacterial, yeast or plant cells, while transfection is a term usually reserved for mammalian cells. Typically the method for transformation of a DNA construct into a host cell is chemical transformation, electroporation or particle bombardment. In chemical transformation, cell are made competent (able to take up exogenous DNA) by treatment with divalent cations such as calcium chloride, which make the bacterial cell wall more permeable to DNA. Heat shock is used to temporarily form pores in the cell membrane, allowing transfer of the exogenous DNA into the cell. In electroporation, a short electrical pulse is used to make the bacterial cell temporarily permeable. Particle bombardment, is typically used for the transformation of plant cells. Gold or tungsten particles are coated with the DNA construct and physically forced into the cell by gene gun.

Molecular Cloning Strategies
PCR
Restriction Digestion
Ligation
Transformation
Transfection

DNA Transformation Protocol

  • Thaw all reagents completely on ice.
  • Add 1 µL of ligation reaction to thawed competent cells.
  • Gently mix by tapping tube of competent cells.
  • Incubate reaction on ice for 30 minutes.
  • Heat shock the competent cell mixture by incubation for 30 to 60 seconds in a 42°C water bath.
  • Incubate tubes on ice for 2 minutes.
  • Add 250 to 500 µL of SOC or LB media.
  • Incubate at 37°C and shake at 250 rpm.
  • Warm selection plates to 37°C.
  • Spread  10, 50, and 100 µL of transformed cells on  selection plates.
  • Incubate plates at 30°C overnight.
Component
Final concentration/amount
Competent cells
to 50 µL
Ligation reaction
1-5 µL
SOC or LB media
950 ml

Transformation Troubleshooting Guide

A successful plasmid transformation is dependent on a number variables including antibiotic concentration, construct size and concentration, and ligation efficiency. Use the troubleshooting guide below to optimize your transformation reactions or get your desired gene in the vector you want the easy way with GenEZ™ ORF Clones. Start with a Search for your gene.

Problem Causes Solutions
Wrong antibiotic was used or antibiotic concentration was too high
  • Ensure the correct antibiotic was applied to plates.
  • Use only concentration recommended by competent cell or antibiotic manufacturer.
Competent cell viability is low
  • Thaw competent cells on ice and use immediately.
  • Check expiration date of cells.
  • Do not re-freeze cells.
  • Do not vortex cells - gently tap to mix.
Few or no colony
transformants
 DNA insert encodes protein that is toxic to cells
  • Use a lower incubation temperature (25–30°C).
  • Use a cell strain and vector designed for tightly controlled transcription.
Heat-shock incubation too long
  • Reduce incubation time from 45 to 25 seconds.
Construct is too big
  • Use electroporation for vectors over 10 kb.
Too much ligation mixture was used for the transformation
  • Ligation reaction components can inhibit transformation.
  • Dilute ligation reaction with TE buffer (up to 5 times).
Too much DNA in reaction
  • Use no more than 1-10 ng of DNA in 5 µl for a 100 µl reaction or in 1-3 µl for a 50 µl reaction.
Low ligation efficiency
  • Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10.
  • Use a DNA concentration of 1-10 µg/ml.
Construct recombined with genomic DNA
  • Switch to a Rec A- cell strain.
No Plasmid in
Colony tranformants
 Antibiotic concentration too low
  • Use antibiotic concentration recommended by manufacturer.
Antibiotic is degraded
  • Aliquot working volumes of antibiotic and avoid freeze-thaw cycles.
  • Add antibiotic to liquid plate media after sufficient cooling.
No insert in colony
tranformants plasmids
 Vector re-ligation
  • Vector insert ratio not optimal. Use a vector:insert molar ratio from 1:1 to 1:10. Use a DNA concentration of 1-10 µg/ml.
  • Dephosphorylate DNA with phosphatase to prevent re-ligation.
Sequencing of tranformants
plasmid reveals wrong
plasmid sequence
 DNA insert encodes protein that is toxic to cells
  • Use a lower incubation temperature (25–30°C).
  • Mutations introduced by initial PCR
  • Use a high-fidelity polymerase.
Inconclusive sequencing artifacts
  • Repeat sequencing reaction.

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