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Some of the plasmids in the “expmt” sample had the gene of interest inserted backwards. How...

Some of the plasmids in the “expmt” sample had the gene of interest inserted backwards. How can “backwards” insertions be avoided altogether in recombinant plasmid production?

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Answer #1

To avoid reverse orientation :-

Design a pair of PCR primers, with one annealling to the vector and one on the insert, in such a way that they would only make the product if the desired orientation is present. You can also design a complementary pair as a control to look at the other orientation. This way, you can perform colony PCR to identify the orientation of your insert straight out of the plate.

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