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Molecular Cloning Center

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.

Molecular cloning strategies
PCR
Restriction digestion
Ligation

Transformation

Transfection

Molecular cloning handbook

Bioinformatics tools

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

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