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Molecular Cloning Central
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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).
  • Use a cell strain and vector designed for tightly controlled transcription.
Mutations introduced by initial PCR
  • Use a high-fidelity polymerase.
Inconclusive sequencing artifacts
  • Repeat sequencing reaction.

Transformation protocol »