The diagram below shows two DNA molecules, a plasmid for...

The diagram below shows two DNA molecules, a plasmid for cloning and an insert - the gene encoding green fluorescent protein (GFP). Your task is to clone GFP into the EcoRI site of the plasmid and analyze the resulting putative clone. Consider scenarios A and B below and answer the questions. A. The insert is oriented in the same direction as the AmpR gene (5'-3'). B. The insert is oriented in the opposite direction as the AmpR gene (3'-5'). EcoRI XhoI EcoRI 800 bp 200 bp Green Fluorescent Protein 121 bp 702 bp 177 bp EcoRI 450 bp AmpR PvuII Plasmid: 3600 bp 1. Under scenario A, what is the result of cutting the recombinant plasmid with EcoRI? [ Select ] 2. Under scenario B, what is the result of cutting the recombinant plasmid with XhoI? [ Select ] 3. Under scenario A, what is the result of cutting the recombinant plasmid with XhoI + PvuII? [ Select ] 4. Under scenario B, what is the result of cutting the recombinant plasmid with XhoI + PvuII? [ Select ] 5. What is the size of the GFP protein, assuming that the coding sequence (including stop codon) is 702 bp? [ Select ]

Transcript

00:01 So you have a diagram showing a dna molecule, a plasmid for cloning for insert.

00:05 The plasmid at 3600 base pair.

00:08 You have an ampicillin resistance site and the arrows show you the direction of the expression.

00:16 And then you have a equar1 and you have a ppu2.

00:21 Between the two sites you have 450 base pair.

00:24 And you also have an insert which is about 1000 base pair long with equar1 site on both sites.

00:32 And there also is a zo1x which is in between the two equar1 sites.

00:39 And then the first e is 800 base pair away from the x and then the x is 200 base pair away from the second e.

00:51 So the question says that consider scenario a and b.

00:57 A is that the insert is in the same orientation as the ampicillin.

01:02 And then the b is the opposite orientation of the ampicillin gene.

01:09 So the question says, a, under scenario a, what is the result of cutting the recombinant plasmid with equar1? so let's take a look of what is scenario a.

01:23 So scenario a is the same direction of ampicillin resistant gene.

01:28 So when you insert that into the plasmid, so you have ampicillin resistant right here.

01:37 And then you have a equar1 and you can see that you have an insertion right here.

01:50 And this is another e right here.

01:52 And the orientation of the gfp is this way, the same direction as ampicillin.

01:59 You can see the arrow.

02:01 So in that case, you insert this 1000 base pair sequence in it.

02:08 So you have a x somewhere here, which is 800 here and 200 here.

02:17 So now the result of cutting recombinant with equar1, you'll cut the fragment out at here and here.

02:35 So you'll produce a fragment, which is the plasmid 3600 base pair.

02:48 And then you have this insert 1000 base pair and both has equar1 sticky end.

03:04 Now b scenario, the opposite orientation.

03:23 So the ampicillin is this way, but the insertion is the other way.

03:29 So the zoe is the recombinant plasmid with equar1 only.

03:49 It's still going to only cut here and here.

03:52 So you'll produce the same pattern of fragments compared to scenario eight, which you'll have a plasmid, which is a 3600 base pair with equar1 sticky end.

04:14 And then you'll have a insertion, the opposite orientation, but still 1000 base pair.

04:24 So you won't be able to tell the scenario a and b by cutting with equar1.

04:32 Now the next question, three, under scenario a, what is the resulting, a result of cutting the recombinant plasmid with zoe and pbu1? so now, so let's go back to scenario a.

04:59 Recombinant 3600 with e here, e there, insert is in the same direction as the ampicillin.

05:18 So the zoe is somewhere here and the pbu is somewhere here.

05:29 Now in that case, if we cut this recombinant plasmid with zoe1 plus pbu1, pbu2.

05:37 So it's going to cut right here and there...

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