What techniques can be used to verify that subcloning has successfully produced the desired plasmid?

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Answer 1

Answer #1

Two techniques routinely used to determine if subcloning has produced the desired plasmid are:

Restriction Digestion: Multiple restriction enzymes are used to determine the relative position of the restriction sites in the insert and the plasmid backbone; it can be determined if the insert is present or absent. The orientation of the insert sequence can also be determined.
Sequencing: Sequencing the plasmid with primers designed to work only when the insert is present and in the correct orientation is another technique often utilized. This allows for not only ensuring that the insert is present, but also that it is free of mutations.

Answer 2

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