Cloning Troubleshooting Guide | Thermo Fisher Scientific

You have worked hard to clone your DNA fragment of interest—you have performed restriction digestion, fragment preparation and purification of the desired insert, vector and insert ligation, bacterial transformation, and finally plating of transformed colonies. As you screen for inserts, you see something amiss blocking your progress.

What do you do? First, start by tracing the workflow in reverse, checking each step for any problem that may have occurred. If proper controls were included in each step, troubleshooting will be much faster and easier.

Learn how to resolve these cloning issues with the following helpful suggestions assembled by our expert team of technical applications scientists.

Sequence errors in insert

Cloning issues

Few or no transformants

Possible cause	Recommendation
Cell plating step
Insufficient cells	Use the volume recommended by the manufacturer to plate transformed cells. Adjust cell dilutions as necessary to obtain the desired number of colonies. Recover the cells in enriched medium (e.g., S.O.C medium) after heat shock and allow sufficient time for growth (e.g., 1 hour) before plating.
Incorrect antibiotic	Make sure that the antibiotic used in the media is the same as the vector’s resistance marker.
Antibiotic concentration is too high	Verify that the correct amount of antibiotic was used in the media.
Bacterial transformation step
Poor transformation efficiency	Check the competency of your cells using 0.1 ng of an intact, supercoiled vector DNA (e.g., pUC19) in a transformation reaction. The competent cells should yield at least 1 x 10⁶ transformants per µg of supercoiled DNA. This corresponds to approximately 100 colonies when using 0.1 ng of uncut plasmid for a transformation. For longer inserts (e.g., >5 kb), consider electroporation instead of chemical transformation to increase transformation efficiency. For chemical transformation, choose competent cells with higher transformation efficiencies (>1 x 109 CFU/μg) or those specifically tested to handle large plasmids. See also transformation troubleshooting guide.
Toxic insert	Some cloned sequences may not be tolerated by the host E. coli strain. Check the target sequence for strong E. coli promoters and for inverted repeats. If the protein expressed by the cloned sequence is toxic to E. coli, try using promoters that have low level expression or that are inducible with tight regulation. Also consider using a low copy number plasmid as a cloning vehicle. Try a different strain that tolerates repeated sequences (e.g., Stbl2 E. coli). Try growing the cells at a lower temperature (30°C or room temperature). Colonies will take longer to appear.
Excess ligase in transformation mixture	The presence of T4 DNA ligase can reduce transformation efficiencies. Do not use more than 5 µL of ligation mixture for 50 µL of competent cells or 1 µL for electrocompetent cells.
Ligation step
Poor ligation efficiency	Perform a control reaction to check that the ligase remains active. Optimize ligation parameters, such as insert:vector ratios, reaction time, and reaction temperature, to maximize insertion of the desired fragment.
Poor DNA quality	Ensure the insert and vector DNA fragments are free of remnants of the digestion reaction (e.g., active restriction enzymes) or contaminants (e.g., excess salts, EDTA, phenol, etc.) that may inhibit the ligation reaction. Purify the vector and insert by gel electrophoresis, column purification, or phenol:chloroform:iso-amyl solvent extraction prior to ligation.
Ligase carried over to transformation mixtures	The presence of T4 DNA ligase can reduce transformation efficiencies. Do not use more ligase than recommended in the ligation reaction. If necessary, inactivate T4 DNA ligase by chloroform extraction, heat inactivation, or spin-column purification before transformation.
Incompatible ends in ligation	Review overhangs generated by the chosen restriction enzymes for compatibility.
Fragment preparation step
Lack of 5′ phosphate	If using dephosphorylated vector, make sure the insert possesses 5′ phosphates for ligation.
Suboptimal blunting	In blunt-end cloning, the overhangs generated by the restriction enzymes may have been inefficiently filled-in and/or trimmed. Use optimal blunt-end methods for the overhangs.
UV-damaged DNA	DNA may have been damaged by UV light during excision from agarose gel. To minimize UV-induced damage, use a long wavelength UV (360 nm) light box when excising DNA from gel and limit exposure time as much as possible. If using a short wavelength (254–312 nm) light box, limit exposure of the DNA to a few seconds and keep the gel on a glass or plastic plate during illumination. Alternatively, use dyes with long excitation (less damaging) wavelengths to visualize the DNA. Another option is to load the DNA sample in two or more lanes. After electrophoresis, cut away and stain only one lane with ethidium bromide. Use this stained lane as a reference for excising the DNA from the unstained lanes.
Restriction digestion step
Unexpected or incomplete cleavage	Ensure restriction enzymes are free of contaminating endonucleases, exonucleases, or phosphatases that may damage the DNA ends. Use enzymes that are produced under highest quality standards for cloning. Perform restriction digestion as recommended by the enzyme supplier. Check for reaction conditions, buffer compatibility, and required cofactors for optimal digestion. Check DNA fragments by gel electrophoresis after restriction digestion to assess the correct band sizes, as well as any unexpected cleavage such as smears and extra bands. Sequence the digested fragments to check the cut sites, if necessary.

Many colonies containing empty vector

Possible cause	Recommendation
Bacterial transformation step
Insert is toxic to the cells	Some cloned sequences may not be tolerated by the host E. coli strain, increasing the background of cells with the empty vector. Check the target sequence for strong E. coli promoters and for inverted repeats. If the protein expressed by the cloned sequence is toxic to E. coli, try using promoters that have low level expression or that are inducible with tight regulation. Also consider using a low copy number plasmid as a cloning vehicle. Try growing the cells at a lower temperature (30°C or room temperature). Colonies will take longer to appear. Try a different strain that tolerates repeated sequences (e.g., Stbl2 E. coli).
Fragment preparation (dephosphorylation) step
Vector self-ligation	To decrease vector self-ligation, dephosphorylate the vector with an appropriate alkaline phosphatase. Ensure alkaline phosphatase is completely inactivated or removed after dephosphorylation. If using dephosphorylated vector, then the dephosphorylation reaction may have been inefficient. Perform a negative control reaction by ligating dephosphorylated vector and transforming into cells. Repeat alkaline phosphatase treatment of vector if necessary.
Restriction digestion step
Incomplete cleavage	Gel-purify the digested vector and insert on an agarose gel to assess cleavage efficiencies. Confirm vector cleavage by using digested, unligated vector in a transformation reaction as one of the reaction controls.

Many colonies without vector

Possible cause	Recommendation
Bacterial plating step
Antibiotic concentration too low	Verify that the correct amount of antibiotic was used.
Antibiotic has degraded	Allow agar to cool to approximately 55°C before adding antibiotic to avoid temperature-induced degradation affecting potency. Some antibiotics (e.g., ampicillin and tetracycline) are sensitive to light. For longer term storage, store plates containing light-sensitive antibiotics away from light. Plate competent cells without transformation as a negative control to check antibiotic selection.
Satellite colonies	Some faster growing strains of E. coli may degrade antibiotics quickly, allowing the formation of smaller non-resistant colonies around transformants called satellite colonies. Avoid overgrowing the cells by incubating less than 16 hours after transformation. Pick well-isolated colonies with no visible satellite cells nearby for screening.
Cell density too high	Avoid over-plating transformed cells which could lead to degradation of antibiotics in the medium and overgrowth of cells without the vector. Use the recommended cell volume and adjust dilutions as necessary to reduce and optimize the number of transformed colonies formed.
Bacterial transformation step
Antibiotic resistant E. coli strain	Check the genotype of the E. coli strain. For example, INV110 are resistant to tetracycline and streptomycin.
Plasmid recombination into the chromosome	Plasmid sequence may have integrated into the bacterial genome, conferring antibiotic resistance to the cells without the vector. Use competent cells with the recA mutation to prevent recombination of DNA and allow stable propagation of the vector.

Incorrect or truncated inserts

Possible cause	Recommendation
Bacterial transformation step
Unstable insert	For cloning of unstable DNA which may contain direct repeats, retroviral or lentiviral sequences, use specifically-designed competent cells for the transformation step to prevent plasmid recombination.
Fragment preparation step
Incorrect band extracted	Ensure the correct band was excised from a well-resolved gel. Use appropriate gel percentage and running voltage for good separation of all fragments that might be present after restriction digestion. Be mindful of bands that may co-migrate during electrophoresis.
Restriction digestion step
Unexpected cleavage	Ensure chosen restriction sites are unique for the target sequence. Check the vector map, map the restriction sites using the vector sequence, or sequence the template as appropriate. Target fragment may be truncated by contaminating exo- and endonucleases present in the cloning enzymes. Use only high-quality enzymes tested for the presence of these contaminants. Check for extra bands and smears in electrophoresis that could indicate the presence of contaminating nucleases. Follow reaction conditions recommended by the enzyme supplier to avoid star activity (i.e., altered specificity) and incomplete digestion. Alternatively, use restriction enzymes that are designed for complete digestion without star activity.

Sequence errors in insert

Possible cause	Recommendation
Bacterial transformation step
Insert mutation	Mutations may have occurred during plasmid propagation in transformed cells. Pick a sufficient number of colonies for sequencing during screening. If all colonies show the same mutation in the insert, it may have originated in the starting template. If the insert is PCR amplicon, use a high-fidelity PCR enzyme to minimize introducing nucleotide errors during amplification. For cloning of unstable DNA which may contain direct repeats, retroviral or lentiviral sequences, use specifically-designed competent cells for the transformation step to prevent plasmid recombination.
Fragment preparation step
UV-damaged DNA	To minimize UV-induced damage, use a long wavelength UV (360 nm) light box when excising DNA from gel and limit exposure time as much as possible. If using a short wavelength (254–312 nm) light box, limit exposure of the DNA to a few seconds and keep gel on a glass or plastic plate during illumination. Alternatively, use dyes with long excitation (less damaging) wavelengths to visualize the DNA.

For more troubleshooting information, please check our Cloning Support Center or contact our technical support team.

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