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Part II. Screening Transformants
Part II
Screening Transformants The screening of transformants can be done in a number of ways. In practice, the techniques used by a scientist will often depend on the reagents, time, and budget available. In the next few experiments, you will be performing some of the more common techniques used to determine whether a bacterial clone carries a gene of interest. While the forced cloning experimental design in the previous experiments has ensured the proper results, these screening methods will also confirm that the egfp gene was inserted in the correct orientation and reading frame to produce the GST::EGFP fusion protein. It is important to confirm that a recombinant molecule to be used for large-scale protein expression comprises exactly what is intended: one copy of the vector and one copy of the insert. The restriction mapping experiment will confirm that an insert of the correct size is present. The PCR screen experiment will determine if the gene is present in the correct orientation with respect to the promoter. The visualization of green fluorescence will reveal whether the recombinant protein is folded correctly. And finally, sequencing the recombinant plasmid DNA of a putative positive clone will confirm that no single nucleotide errors were incorporated during the PCR amplification of the egfp gene during cloning. It is important to know and understand a wide variety of screening techniques to troubleshoot if any problems occur during the cloning process.
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Screening Transformants The screening of transformants can be done in a number of ways. In practice, the techniques used by a scientist will often depend on the reagents, time, and budget available. In the next few experiments, you will be performing some of the more common techniques used to determine whether a bacterial clone carries a gene of interest. While the forced cloning experimental design in the previous experiments has ensured the proper results, these screening methods will also confirm that the egfp gene was inserted in the correct orientation and reading frame to produce the GST::EGFP fusion protein. It is important to confirm that a recombinant molecule to be used for large-scale protein expression comprises exactly what is intended: one copy of the vector and one copy of the insert. The restriction mapping experiment will confirm that an insert of the correct size is present. The PCR screen experiment will determine if the gene is present in the correct orientation with respect to the promoter. The visualization of green fluorescence will reveal whether the recombinant protein is folded correctly. And finally, sequencing the recombinant plasmid DNA of a putative positive clone will confirm that no single nucleotide errors were incorporated during the PCR amplification of the egfp gene during cloning. It is important to know and understand a wide variety of screening techniques to troubleshoot if any problems occur during the cloning process.
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