Transformation is mainly performed in molecular cloning to maintain and propagate DNA sequences of interest in bacteria or other host cells. The DNA of interest is incorporated into plasmids or other vectors, which act as a vehicle to shuttle DNA of interest into host cells. The transformed colonies are analyzed after transformation to ensure propagation of the DNA insert. Some of the issues observed include absence of transformants, transformants with incorrect inserts or absence of inserts.
This article on bacterial transformation troubleshooting addresses some common problems and recommendations on how to solve them.
After overnight incubation following transformation, either no colonies or very few colonies are observed on the selective LB agar plate.
|Possible causes for very few or no transformants
|Recommendations to optimize transformation and colony formation
|Suboptimal transformation efficiency
To ensure good transformation efficiencies:
Suboptimal quality and/or quantity of transforming DNA
Cloned DNA or protein that is toxic to the cells
Incorrect strain used to propagate a vector carrying a lethal gene for selection
|Insufficient number of cells plated
Suboptimal growth time and temperature
Incorrect antibiotic or concentration in plates
Improper use of cell-spreading tools
After colony selection and analysis, vector contains an incorrect or truncated DNA fragment as identified by sequencing or restriction analysis (fragment size identified in the gel).
|Possible cause for transformants with incorrect or truncated DNA inserts
|Recommendations to maximize propagation of correct DNA inserts
|Cloned fragment truncated
After colony selection and analysis, vector is empty.
|Cloned DNA or protein that is toxic to the cells
|Improper colony selection method
|Issues in upstream cloning steps
Picked colonies do not grow in a selective media or after colony selection no DNA vector could be obtained.
|Recommendation to improve chances of transformation
|Vector plasmid recombined into the host chromosome
|High number of cells plated
After overnight incubation, too many transformed E. coli colonies are observed making it difficult to pick single colonies (they might even fuse together). Or colonies are spread unevenly forming isolated clusters.
|Possible causes for poor colony formation
|Recommendations to avoid overgrown and clumped colonies
Large number of cells plated
It takes unusually longer to grow cells in liquid media or purified DNA yields are not sufficient.
|Recommendations to optimize cell growth and improve DNA
Improper growth conditions or old colony
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