How to clone with IDT Gene Fragments

We understand how frustrating it is to not obtain any colonies when cloning or not obtain the correct construct while screening. Although IDT Gene Fragments are synthesized using the highest fidelity synthesis methods it is important to acknowledge that mutations can occur at various steps during the cloning process. Design considerations for your gBlocks™ Gene Fragments should be weighed when using a specific cloning method. Although cloning experiments can be complex, here are a couple of troubleshooting steps to consider: 

• Check the design of your IDT Gene Fragment: The gBlocks Order Entry page has a built-in complexity screener that will help you identify potentially troublesome elements, such as repeats, hairpins, and GC fluctuations. You can use the information provided by the tool to help design and remove complexities to facilitate a higher chance of synthesis success.
  
  
• Confirm the concentration of your insert and the ratio of insert to vector: Ensure that the concentration of your IDT Gene Fragment is within the optimal range. If the concentration is too low, it may result in a lack of visible colonies. Also ensure that the ratio of insert to vector is correct.
  
  
• Sequence more colonies. It’s advisable to pick 3 or 4 colonies to screen. This provides the highest probability of ensuring that the recombinant vector is present in the cell and the DNA fragment was inserted in the correct location and orientation within the vector. See Table 1 for an estimated number of colonies to screen. 

Table 1. The approximate number of colonies to screen for a 90% chance of getting a correct clone.

See this DECODED article, Tips for working with IDT Gene Fragments, for how to resuspend, quantify, and calculate copy number.

Why do mutations occur when cloning?  

The cloning process involves multiple steps, ranging from generating recombinant DNA and vector constructs to introducing them into host cells [1]. While cloning experiments can be complex, here a couple of considerations:

Mutations are not dependent on PCR amplification of the IDT Gene Fragment. It’s worth noting that mutations can be observed even without amplifying the insert. PCR amplification of gene fragments prior to cloning should be avoided due to the possibility of introducing point mutations, base insertions, or deletions, which can lead to non-functional or altered gene sequences. Moreover, there are various DNA and cellular mechanisms that can introduce errors  [2]:

• DNA replication can lead to mutations. DNA polymerases are responsible for synthesizing new DNA strands and are not infallible. This can lead to incorrect nucleotides being incorporated into the new DNA strand.
  
  
• DNA repair mechanisms are not perfect. The host cells possess DNA repair mechanisms that fix errors and can also damage the IDT Gene Fragment by introducing mutations during the repair process.
  
  
• Altered recombination events between the IDT Gene Fragment (or DNA insert) and host genome. Additional elements such as plasmids, integrons, transposons, or short DNA sequences can alter recombination events thus leading to mutations  [3].

• Bacterial replication is not perfect and can introduce errors and DNA damage due to various endogenous or exogenous factors:
  
  • The replication speed of host cells. Bacterial replication occurs rapidly to ensure efficient cell division and proliferation [3]. This can increase the likelihood of errors. The faster the replication process, the less time there is for accurate proofreading and correction of errors.
  • Environmental stressors affecting replication mechanisms or leading to DNA damage. Bacteria (host cells) can encounter various environmental stressors, such as exposure to oxidative damage, mutagens, chemical or physical stressors, base analogs, flat aromatic compounds, alkylating or deaminating agents [3]. These factors can compromise the replication process or increase the likelihood of errors causing damage to the DNA.

• Sequence complexity and toxicity can contribute to errors.
  • Sequence complexity: When designing your IDT Gene Fragments, keep in mind that complex sequences, repetitive elements, secondary structures, or regions with extremely high or low GC content can pose challenges for accurate replication. IDT developed the SciTools™ suite of web tools which can help you assess your DNA sequences for complexities and optimize codon usage based on a host organism. See this DECODED article, Codon optimization tool makes synthetic gene design easy, for IDT’s free online Codon Optimization Tool to simplify designing your IDT Gene Fragments.
  • Toxicity: Some gene sequences or inserts can be toxic to the host cells, which can interfere with the normal cellular processes or cause DNA damage. If the DNA insert is toxic, the host cell may exhibit cellular stress responses or undergo genetic changes to cope with the toxicity [2]. These changes can include mutations that occur because of the cellular response to the toxic insert.

  In addition to the factors mentioned earlier, there are several other common mistakes that can contribute to poor results or failed cloning experiments. Here are some suggestions to follow when performing your experiments:

  • Avoid contamination. Here are common contaminants that can affect your cloning experiments: 

  • Foreign DNA contamination: This form of contamination can occur when DNA from unintended sources, such as other plasmid DNA, genomic DNA, or residual DNA are inadvertently introduced into the cloning experiment [3].
    
    
  • Microbial contamination: Bacterial or fungal contamination can occur during the cultivation of host cells or the handling of plasmids or other DNA constructs. Microbial contaminants can affect the growth of the transformed cells or alter the integrity of the cloned DNA [3].
    
    
  • Contaminated reagents: Errors can occur if the reagents used in the cloning protocol such as restriction enzymes, ligases, polymerases, or buffers are contaminated with DNA or other impurities [4].

   

• Minimize freeze-thaw cycles. This can cause physical stress to the DNA which can potentially lead to DNA strand breaks or structural alteration [2]. Follow best practices for storing your IDT Gene Fragments, enzymes, and critical reagents, used in your cloning protocol. 

  If there are few or no colonies after transformation, here are some factors to consider: 

  • Low or inefficient transformation. To validate the experimental conditions and ensure that the transformation step is working properly, include a positive control. This involves using a known, well-characterized DNA sample that is expected to yield colonies. This will help to rule out any issues with competent cells and antibiotic selection. If the positive control fails to yield colonies, it indicates a problem with the experimental setup or transformation process that needs to be addressed.
  • The gene product is toxic to bacterial cells. If the gene product is toxic to bacterial cells, the sequence may spontaneously mutate to allow survival of the cells. An alternative solution would be to check if the gene encoded in the DNA sequence is toxic to the competent cells. If so, use a different competent cell line. If the gene encoded in the fragment is toxic to the host cells, try a different competent cell line or a low copy plasmid. Low copies of the plasmid will undergo fewer replication cycles, generating less protein and lower toxicity in the process.
    
    
  • If the same mutation is across all clones. Even with a median error rate of 1:5000, gBlocks and eBlocks are not sequence-verified; therefore, observing the same mutations across all clones may indicate an error during fragment synthesis. For sequence confirmation, you may want to consider ordering your insert as gBlocks HiFi Gene Fragments, which are NGS-verified, have a median error rate of less than 1:12000, and are manufactured with the same industry-leading, high fidelity synthesis chemistries developed for our Ultramer™ DNA oligos.

  Download our free Cloning Guide, to read more about common methods and important sequence design consideration when designing your experiments.