Need genetics professor or wiz on PCR and Inverse PCR! Can you answer my teacher

Question

Need genetics professor or wiz on PCR and Inverse PCR! Can you answer my teachers comments?

The inverse PCR process we are doing in class has multiple steps which are digesting the isolated DNA from our fly line, denaturing the restriction enzyme, ligating the fragments into circular DNA, annealing the primers facing outward toward the inverted repeats, and synthesizing/extending the copies of DNA (Mistry, 2017). In digestion, we use restriction enzymes that have recognition sites at either end of the engineered EP element and will allow cleavage before the inverted terminal repeats. After the cleavage we will denature the restriction enzymes and will no longer cut at multiple potential recognition sites. Then we will ligate the pieces back together which will create circular DNA. After we will use inverse PCR with the purified piece of DNA, so in this case we will have primers facing outwards and allow the primers to anneal. Then the extension/synthesis of DNA occurs outwards to make cDNA amplify the flanking inverted repeats shown in Figure 4.

Explanation / Answer

Cleavage before inverted repeats is important:-

Creation of circular DNA is important:-

We get primers facing outwards:-

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