How would you confirm that the PCR amplified gene fragment is aligned in the direction of Promoter--> ATG--> --> --> TAA reading frame to successfully express the protein?
The gene that code for the proteins usually contain the Open reading frames (ORF). ORF is the codons series that specify sequence of the amino acid of the protein. ORFs consists the series of the codon that will specify the amino acid sequence of the protein for which the gene codes. The beginning of the ORF occurs with the initiation codon that is ATG usually and ends with the termination codon that are TAA, TAG, or TGA. By searching for these DNA sequences for ORFs will help in telling the correct PCR amplification. This can be achieved by tagging the fluorescent tag for these sequences.
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