I have designed primers for side directed mutagenesis for bac plasmid for deletion of Kpn-I site and transformed it in DH5 cell but no colonies why?

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Noor Rajeeb

If you have designed primers for site-directed mutagenesis to delete a specific restriction site (in this case, the Kpn-I site) in a BAC (Bacterial Artificial Chromosome) plasmid and you are not observing any colonies after transformation, there could be several reasons for this outcome. Here are some potential explanations:

Primer design issues: It's possible that there may be issues with the primer design. Ensure that the primers are designed correctly, targeting the desired site for deletion without introducing any unintended mutations. Verify the primer sequences, including their length, Tm (melting temperature), and absence of secondary structures or hairpins that could hinder primer binding.

Transformation efficiency: The absence of colonies could be due to low transformation efficiency. Check the competency of your DH5α cells to ensure they are competent for successful transformation. Consider using freshly prepared competent cells or optimizing the transformation protocol to improve efficiency.

Toxicity of the modified BAC: The deletion of the Kpn-I site may have unintended consequences, such as affecting the stability or functionality of the BAC plasmid. The modification could render the BAC plasmid toxic to the host cells, resulting in cell death and no colony formation. Consider verifying the compatibility and viability of the modified BAC with the host strain.

Antibiotic selection: Confirm that the appropriate antibiotic resistance marker is present in the BAC plasmid and that the antibiotic concentration used for selection is appropriate. Ensure that the antibiotic resistance gene is functional and active in the host strain.

Screening conditions: If you are using selective media or agar plates for screening colonies, double-check that the medium composition and conditions (e.g., antibiotic concentration, incubation temperature, and duration) are suitable for the growth and selection of transformed cells.

DNA quality and quantity: Assess the quality and quantity of the BAC plasmid DNA used for transformation. Ensure that the DNA is free from contaminants or degradation that could interfere with the transformation process.

It is recommended to carefully review your experimental protocol, primer design, and troubleshooting steps. Additionally, consulting with experienced colleagues or seeking guidance from experts in BAC cloning and mutagenesis could be valuable in identifying the specific issue and optimizing your experimental approach.

Karol Szafranski

The protocol of site-directed mutagenesis uses PCR to generate a full copy of the cyclic vector, typically ca. 5 kbp in size. How large is your BAC plasmid? If it's significantly larger than 5-8 kb it requires adaptation of the PCR protocol to ensure completion of the plasmid within a PCR cycle.

Alternatively, you might refer to protocols of synthetic genomics in order to achieve in vitro cloning of the (mutated) BAC.