I want to insert my GOI in the MCS region of p28a vector, it has his tag so screening is very easy for me. But my GOI has stop codon at the end. So, definitely it will stop the expression of his tag. So, I want to remove the stop codon in my GOI. So, can you suggest me any protocol to remove this stop codon.
Through PCR amplification of your gene of interest; design reverse primer such that it hybridizes with the last 18-21 nucleotides (of your gene) except the ones (three) that form the stop codon (TGA, TAA, TAG). In oligos, you'll include appropriate restriction enzyme sites that are compatible with pET28a vector for directional cloning.
Dear Colleague,
I hope this message finds you well. Removing the stop codon at the end of an open reading frame (ORF) in your nucleotide sequence can be achieved using site-directed mutagenesis. Below is a detailed and logical protocol to guide you through the process.
Protocol for Removing the Stop Codon Using Site-Directed Mutagenesis
Materials
Primer Design
- Forward Primer: Should be designed to anneal to a sequence upstream of the stop codon. It does not need to introduce any changes.
- Reverse Primer: Should be designed to anneal downstream of the stop codon and include the sequence change that removes the stop codon. Ensure that the primer overlaps the region where the stop codon is located and introduces the desired nucleotide changes to remove the stop codon.
Example: If the stop codon is TAA, and you want to replace it with another codon, the reverse primer should include the sequence without TAA, ensuring proper annealing and extension during PCR.
Protocol
Tips for Success
- Primer Design: Ensure primers are specific and have a melting temperature (Tm) appropriate for the PCR conditions.
- PCR Optimization: Optimize annealing temperature and extension time based on the length and GC content of the template.
- DpnI Digestion: Ensure complete digestion of the parental template to avoid background colonies.
- Sequencing: Always sequence the final construct to confirm the desired mutation and the absence of any unintended changes.
By following this protocol, you can efficiently remove the stop codon at the end of the ORF in your nucleotide sequence, allowing for downstream modifications or extensions as needed.
This list of protocols might help us better address the issue.
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