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(asent Princip us ot 8NYU Class0 = Principles ol Bit-157524012eefltoo18c67dc67-771f-496c-9e23 8(2a28ca5r99/js /deliverylbeginTakingAssessn newclasses nyu ~edulporta...

Question

(asent Princip us ot 8NYU Class0 = Principles ol Bit-157524012eefltoo18c67dc67-771f-496c-9e23 8(2a28ca5r99/js /deliverylbeginTakingAssessn newclasses nyu ~edulportalsite/cd378193-70f6-4c7f-90geJClassesPeeetSelecioPointsQuostion 59Multiple cloning site (MCS} dofinedtha plasmid Hhiure nol protont any "hero elgoFitd (or Mony rostrictlon enzymes contoini site within the plasmid which cenor oniy Ont rgalrotlon uymG tha elle contlining Munycloning many unaatia togatnorAlla Iat Many roetriction on

(asent Princip us ot 8 NYU Class0 = Principles ol Bit -157524012eefltoo18c67dc67-771f-496c-9e23 8(2a28ca5r99/js /deliverylbeginTakingAssessn newclasses nyu ~edulportalsite/cd378193-70f6-4c7f-90ge JClasses PeeetSelecio Points Quostion 59 Multiple cloning site (MCS} dofined tha plasmid Hhiure nol protont any "hero elgo Fitd (or Mony rostrictlon enzymes contoini site within the plasmid which cenor oniy Ont rgalrotlon uymG tha elle contlining Muny cloning many unaatia togatnor Alla Iat Many roetriction onzymas 'plnemid which containa Mthln Bobet Sdleciod Hninia Oleatlon 60 0t 80



Answers

DNA Cloning The plasmid cloning vector $\mathrm{pBR} 322$ (see Fig. $9-3$ ) is cleaved with the restriction endonuclease $P$ stl. An isolated DNA fragment from a cukaryotic genome (also produced by PstI cleavage) is added to the prepared vector and ligated. The mixture of ligated DNAs is then used to transform bacteria, and plasmid-containing bacteria are sclected by growth in the presence of tetracycline. (a) In addition to the desired recombinant plasmid, what other types of plasmids might be found among the transformed bacteria that are tetracycline-resistant? How can the types be distinguished? (b) The cloned DNA fragment is 1,000 bp long and has an EcoRI site 250 bp from one cnd. Three different recombinant plasmids are cleaved with EcoRI and analyzed by gel electrophoresis, giving the patterns shown below. What does each pattern say about the cloned DNA? Note that in $\mathrm{pBR} 322$, the Pst and EcoRI restriction sites are about $750 \mathrm{bp}$ apart. The entire plasmid with no cloned insert is 4,361 bp. Size markers in lane 4 have the number of nucleotides noted. FIGURE CANT COPY

Mhm. Sorry, specific. Mhm. Inversion. All stable? Yeah. Yeah. Yeah. Mhm. All stable. Mhm. Stupid. Right. Naturally occurring DNA plasma contain a pair of long non tend um and what it repeats that under both frequent. Mhm. Yeah. I sleep local. Okay. Rec combination. Mhm. Mhm. Right. two plasma inversion consumers? I don't know. Yeah. Okay. Are you so much that exists in the cell in equal number? Mhm. Right. In the two you circle plasma? Mhm. Mhm. Last week of s Kerry was I. Mhm. Such inversion is catalyzed. Okay. Yeah. Yeah. Next. No. Right. Last week. Okay, encoded. Mhm sites. Mhm specific. Mhm. The companies, Yeah. Yeah.

Cool. We're going to answer a question on recombinant DNA. What we're expected to do is they are expected to give in the form of arrange the steps of the common in DNA technology in order. So we have been given a few options in that. So the first step always in recombinant DNA technology is digestion, division restriction, digestion. So the first thing that we do is digest director and foreign DNA. Is this any destruction anxiety here in this question? It is equal to one which then activates the antibiotic resistant steam dr Saperstein. So this in activates the A. B. R. Team. That is the first step. First step this restriction decision. So the second step would be and the first step is in the fourth please in order of the question. So that's about it. Now the second question after digestion is ligation. The second step after digestion as ligation. So we are going to like it the digested DNA. Together with the foreign DNA. Now you have a foreign DNA. And digested DNA. And what we're gonna do is they're they're cut now there restriction that they are digested. But now what you're gonna do is the next step is you're going to catch them. You basically joined them and to do that. You need you need like you need to like get them. That process is known as ligation. It is done by like gazes. They gave us a enzyme responsible for ligation. They are known as molecular glue. Yeah just known as destruction conditions. Uh The restriction enzymes are known a known as molecular scissors. And like gazes are known as molecular view. So the next step is negation. We like it the devices to D. N. A. And the warranty in it together. And that in the fifth step in the question the next what we do after digestion and legation, we transformed the competent cells. This is where the real actual transformation has and this step is known as just transformation. The DNA is transformed so the competences are transformed and that is the first step in the question. So 451 This is the order that the goal in restriction, tradition ligation and transformation. The next step our what is is to select the lack of an diverted resistance. See that is the first two. So that would be second step in the west and next what you do is select the blast mitt, antibiotic resistance. Street richard ST. This is the now so now this is the order in which is which it is going to be the recumbent in the unit. Technology happens in this order 45123 As I've already said the steps, so this is going to be it and feel that which option suits the answer that we have found. The answer would be option eight. So the correct answer is optioning. And the order in which the recombinant DNA the steps and recombinant DNA technology is 4512 and three from the destruction decision to this election of the colonies and letting the protein group or the bacteria divide and recovering the proteins from it from here today. So that is how it is going to be these other steps involved in re common in DNA technology. And the correct answer is optioning.

Hi, everyone. My name is Eric. And let's review problem 47. So in this question, they're asking us to describe the process of molecular cloning. Now, molecular cloning is the process where you take your DNA of interest, you insert it in a plasmid, and then you're going to place it in a host to replicate and produce mawr amounts of your DNA of interest. And the process to do that is actually what we're looking for here. So we're looking at four different choices A, B, C and D, and I'll be walking us through what is the correct answer and why. The other answers are not correct. So the first answer is letter A. So let's start with a So they're saying that the foreign DNA and the plasma are cut with the same restriction enzyme, and the DNA is inserted into a gene. So let's start there. So our plasma is a circular piece of DNA, and it's mostly common found inthe e bacteria. So with the plasmid, we're going to cut at specific sites with the restriction enzyme, and that's going to allow us to insert our foreign DNA into the same spot and in the problem. They described it as cutting into the lack Z gene. So by cutting into the lack see Jean, we're actually going to be disrupting this gene. And the legacy gene is responsible for metabolizing lactose. So lactose is a molecule, and Black sea is the gene that allows the host to break down and utilize lactose. So that would be the first part where we're going to be inserting our DNA of interest inside the laxity gene. All right, and then did said that the DNA and the vector are a kneeling, so then that would allow us to make a new plasmid. But instead our DNA of interest will be bonded in the spot where the Lexie should be, all right. And then this plasma is going to be transferred into a bacterial host, and it's going to be selected for using ampicillin, right? So somewhere on this plasmid will be an amp resistant jean, so that allows the bacteria to survive on episode. And so that's how we know if the plasma has been taken up into the bacteria. The ones that survive on AMP Resistance is what we're looking for, and once we have it, into a bacterial host. We're going to be able to produce more plasmids. I mean, with our DNA of interest. So it's going to replicate and produce our plasma with our DNA of interest and how we can tell that our DNA has been taken up into the correct spot is that if we introduce what is called X gal X gal is similar to lactose, so X gal is actually the artificial version of black toas. So by introducing X gal, if the bacteria cannot metabolized X gal, then that means that our bacteria have successfully taken up before and DNA and therefore our molecular cloning is successful. So part a following those steps is true. The answer would be part egg. But to further explain, let's look at B, c and D. So and be already the first step is the nature ring using high heat, so that is not correct. We don't de nature. We have to restriction enzyme cut first, so be is not an answer. Let's look at sea, so see they're going to cut. Okay, so that's a good part. They're starting with cutting, and they inserted into the plasmid. Then they allowed to kneel. But then the thing is here, is that they They said at the end here that it shows an inability to synthesize X gal. We're not synthesizing X gal. We're introducing X gal into the environment. We're giving it X gal so that we can see if it metabolizes it or not. So because we're not synthesizing X gal, see is not the correct answer either. And finally, let's look at D. So in part D, they're saying that they want to introduce it into a viral vector. And that is not part of molecular cloning. Because typically, we're going to be focusing it in a bacterial host, right? And additionally, we're not synthesizing X gal. So that is also another reason why we're not picking answer D. So the process of molecular cloning the answer is a thanks for watching. And I'll see you next time. Bye bye

Well, this question. We're going right back to the blue white screen technique. In fact, that's the entire answer to this question, and we could just stop right there. But of course, I want to give us more information about so Geneticist uses a placement for cloning. There's the legs E jean energy. New confers resistance to penicillin Again Sound likely wife of tring. Remember, you attach a gene that has resistance penicillin, and you use X delectable. Glad today's How are you going to Genesis? Inserts a piece of for Indiana into a restriction site is located within the legs region. Remember, because you put it in between the gene to disrupt its function that way. You know if the gene is present, you have energy isn't present. You have function and use the plasma transported back to you. So identify or explain how the genesis unidentified bacteria that contains a copy of the placement with its foreign. So basically, what the Dennis's needs to do. I used to go out there and do its blue screenplay. Admit that they remember the blues room, how you make it method. You get two different types of bacteria, you get the usually white, which white marks? So that was great. And so you played the bacteria on argon medium containing Kinsella. So the media seven penicillin and this helps to select for sales. There's taking a replacement. The medium also should have X gal on it, which induces originally was actually about ways because they induces the lack operas. So sales have taken up the placement without the for Indiana. So they're taking the place, but they need to get the foreign united you won't is gonna produce a functional beta go back two days and make a blue dye sales. They have taken up the placement looked for Indiana is gonna make a white guy because you insert it the gene in place to disrupt the function of Exhale.


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