Resources »
Technical Resource Centers »
Gene Technical Resources »
Molecular Cloning Central »
Transformation Troubleshooting Guide
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 |
| |
Competent cell viability is low |
| |
Few or no colony transformants | DNA insert encodes protein that is toxic to cells |
|
Heat-shock incubation too long |
| |
Construct is too big |
| |
Too much ligation mixture was used for the transformation | Ligation reaction components can inhibit transformation.
| |
Too much DNA in reaction |
| |
Low ligation efficiency |
| |
Construct recombined with genomic DNA |
| |
No Plasmid in Colony tranformants | Antibiotic concentration too low |
|
Antibiotic is degraded |
| |
No insert in colony tranformants plasmids | Vector re-ligation |
|
Sequencing of tranformants plasmid reveals wrong plasmid sequence | DNA insert encodes protein that is toxic to cells |
|
Mutations introduced by initial PCR |
| |
Inconclusive sequencing artifacts |
|