Locations/positions in the DNA of cuts for PGLO...

Locations/positions in the DNA of cuts for PGLO digested with EcoRI & HindIII EcoRI 2063 bp Hindill 2114 bp HindIII 1465 bp Plasmid = 5371 bp list out the resulting fragment sizes after the DNA digest. For example, in column 1 we are looking at the resulting fragment sizes when the PGLO plasmid is digested (cut) by the Enzyme Pairs EcoRI & Hindill EcoRI & Hindill restriction indIII & EcoRI & EcoRI & enzymes EcoRI amHI BamHI BamHI PGLO PAMP and HindIII. AMP PGLO PAMP Fragment 4722bp sizes (bp) 598 bp 51 bp Based on the plasmid DNA sequence and restriction sites, we predict to see three fragments of size 4722, 598, Total #bp 5371 bp in plasmid Fragments smaller than 400 bp very hard to see on the gels that we use and because of the low concentration of plasmid that we use in the analysis.

Transcript

00:01 So you have a circular plasmid pmbbs.

00:07 So the plasmid is being digested with different restriction enzymes, which recognize the dna of a specific sequence, all we call restriction site.

00:17 So what we are looking at is a geol electrophoresisphysis photo.

00:21 You have seven lanes, well, actually eight lanes.

00:25 The first one is the undigested lane, which was the minus sign.

00:32 And the next three lane will be the single digestion with only one enzyme.

00:38 So the second lane is equiline, equine, equal one only.

00:42 The third lane is bamatri -1 digestion only.

00:46 And the fourth lane is zoh -1 digestion only.

00:50 The next two lane will be double digestion, which means two enzymes are being used.

00:55 So, for example, the fifth lane is the double digestion of equate -1 and bamatru -1.

01:02 And then the sixth one is double digestion of equatechre one and zohin.

01:07 And the last one is the double digestion of bavich 1 and zohan.

01:11 And the last one is the latter of the dna.

01:13 It gives you the size guide.

01:16 So if you look at a band in the previous lens, you're able to tell the size of the bands according to the guideline of the latter.

01:25 All right.

01:26 So now we know which sample has what enzyme.

01:31 So let's take a look at all these questions.

01:34 So the first thing says, what is the total size of this pmbbs plasmid in base period? now, the first lane here is undigested.

01:43 So we saw you.

01:45 So the undigested circular dna is going to actually run in the super coil structure, which is the most comfortable confirmation of dna in agorose gel.

01:56 So it's not a circular, it's actually a super coil structure.

01:58 So the super coil structure runs a little little faster than its actual size.

02:03 So you can see that you have two little bands in an undigested lane.

02:08 So it's hard for you to tell what is the actual size of the plasmid.

02:15 But if you look at the next three lanes with single digestion, itquare 1, babbage 1, and zoh 1, each of these three lanes will give you the same band.

02:29 And if we look at the size, these re -band is around 3 ,000 base pair.

02:38 So this is actually a single digestion, the restriction enzyme cut actually linearized the circular plasmid dna.

02:49 And the linearized dna actually reflect the actual size of the plasmid.

02:54 Now we can tell that the total size of the plasmid, pmbbs, is actually about three.

03:02 Thousand base pair long.

03:04 So as you can see, the supercoiled dna is being cut by restriction enzymes once and then basically it become linearized.

03:17 So this actually equals 3 ,000 base pair.

03:22 So the first question, p mbbs has a size of 3 ,000 base pair because again this is a linear fragment, which will reflect the actual size or base pair of the plasmid.

03:43 So that's question number one.

03:45 So remember in the future, if you want to tell a size of a plasmid, you're going to have to use a single cut enzyme to linearize the circular dna.

03:56 So you can know for sure the size of the plasmid.

04:01 The second question, how many cut sites on pmbbs plasmid does each r? our restriction enzyme have.

04:10 So according to our prediction, you can see that each of these enzyme cut the plasmid once.

04:19 And that is why all three enzymes were produced the same band, which is 3 ,000 base pair.

04:27 So how many cut sites in the plasmid does each r &r have? so our question, our answer is, it doesn't matter which kind, bamach 1, zoh1, and e...

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