Restriction enzymes question

[dli42395]

So there's a question on a practice exam that basically asks that if you use EcoRV as an restriction endonuclease, and you create a recombinant plasmid vector with a plasmid that has one EcoRV site, how many bands appear when you perform PCR.

Then answer given is 2 bands, since it is basically cutting a strand of DNA into 2 strands. But this confused me because I thought plasmids were circular, so if you if you only have one restriction site, wouldn't you pretty much just open up the plasmid but it would still be a single strand?

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[doctor.octopus]

When you insert a DNA segment into a plasmid, you first isolate the DNA segment you want to insert, using restriction sites at either end of the desired sequence. Then you use a restriction enzyme to open the plasmid, and then ligate the desired segment into the plasmid.

The DNA insert and the opened plasmid must then have sticky ends that are complimentary; if not, you need to add primers onto the ends of the DNA insert. These primers would need to contain the EcoRV restriction sequence; you would then need to apply the EcoRV restriction enzyme, to result in a DNA insert that has sticky ends that can complex with the sticky ends in the plasmid.

This means that, after you've inserted it, you now have functionally 2 EcoRV sites -- one on either end of the DNA insert, giving you 2 bands on your PCR. One would represent the plasmid, the other would represent your DNA insert.

Let me know if this didn't answer your question

 

[doctor.octopus]

here's a relevant figure -- the ? is your DNA that you inserted using only EcoRI.

 

[dli42395]

When you insert a DNA segment into a plasmid, you first isolate the DNA segment you want to insert, using restriction sites at either end of the desired sequence. Then you use a restriction enzyme to open the plasmid, and then ligate the desired segment into the plasmid.

The DNA insert and the opened plasmid must then have sticky ends that are complimentary; if not, you need to add primers onto the ends of the DNA insert. These primers would need to contain the EcoRV restriction sequence; you would then need to apply the EcoRV restriction enzyme, to result in a DNA insert that has sticky ends that can complex with the sticky ends in the plasmid.

This means that, after you've inserted it, you now have functionally 2 EcoRV sites -- one on either end of the DNA insert, giving you 2 bands on your PCR. One would represent the plasmid, the other would represent your DNA insert.

Let me know if this didn't answer your question

Ok, that makes sense. Thank you