In this experiment, we will isolate plasmid DNA from E. coli cells that potentia

Question

In this experiment, we will isolate plasmid DNA from E. coli cells that potentially harbor a pJET plasmid with your unknown gene inserted into the MCS.? Describe what you would expect to see on an agarose gel under the following conditions after restriction digestion with NdeI and XhoI:

1. The plasmid does contain your gene of interest flanked by the XhoI and NdeI sites

2. The plasmid contains your gene of interest and the XhoI site, but the NdeI site has been lost. (Hint: there are two possible results)

3. The plasmid does not contain your PCR product at all.

Explanation / Answer

1. If the plasmid contains the insert with the desired restriction sites, restriction digestion will release the insert from the construct. So, there will be two bands on the agarose gel. One corresponding to the linearized vector and the other corresponding to the insert.

2. If the plasmid contains only Xho I sites
i. If it contains a single Xho I site, the construct will be linearized upon Xho I digestion. So, there will be a single band corresponding to the linearized construct (Vector + insert)
ii. If it contains two Xho I sites flanking the insert, there will be two bands.

3. If there is no insert, Xho I digestion linearizes the vector assuming that the vector has Xho I site. If the vector does not contain Xho I site, there will be supercoiled plasmid in the gel.

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