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November 28, 2018 at 1:12 PM
In order to insert a human gene into a plasmid, both must be cut with an enzyme.
What enzyme forms covalent bonds between restriction fragments?
This is a DNA fingerprint exhibiting samples from the victim, two suspects, and the crime scene. Which of these DNA fragments are common to both the victim and Suspect 2?
After introducing recombinant plasmids into bacteria, the next step toward producing clones of the desired human gene would be which of the following?
10 – A DNA sequence that can be read the same backward and forward is called a palindrome.
11 – What is a DNA library? Draw a simple flow chart to show how you would proceed to prepare a DNA library of a certain strain of bacteria.
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01:00
1. You want to clone human DNA sequence into plasmid. This DNA fragment was cut out of the human genome with restriction enzyme Sunl, whose restriction site is shown in the figure below:Sunl restriction siteCGTACG GCATCCPlasmid cloning region ACGATCCCGAGTKSTACACGTGGTACCACAATTCCTTCGTACCTTTAAAACA TCCTACEGCTCACATOTGCACCATCGTCTTINGGAACCATGGRAAITITGT Bamh Kori EcoRi Asp7101 DrelThis figure also shows the multiple cloning region of your plasmid, with the potential restriction sites marked: Which of these restriction enzymes could be used to cleave the plasmid for successful insertion of this human DNA fragment? Note that there could be more than one correct answer. B. Briefly explain how you would go about cloning the fragment into the plasmid. C. You successfully clone the human DNA into the plasmid and store it in the freezer. Several months later, your advisor asks you to use this recombinant plasmid to prepare a large quantity of the human insert sequence with as little plasmid sequ…
01:14
1. We can construct our gene library by cloning the PstI fragments into either plasmid pBR322 or phage lambda. Which one should we choose and why? 2. Suppose we want to insert the PstI fragments into the Bam HI site within the gene for tetracycline resistance (tetr) in plasmid pBR322. If both of these restriction enzymes produce cohesive ends, how can this be done? 3. After transforming E. coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing DNA insert)? 4. If a million tetracycline-sensitive, ampicillin-resistance clones are grown on nutrient agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P? 5. After selecting a clone carrying the gene for protein P, its cells are propagated to high density in nutrient broth. The chimeric plasmid (with an insert of the gene for protein P) is then extracted and purified from the rest of the cellular DNA. How can we now isolate the gene from t…
02:12
RECOMBINANT DNA: PLASMID VECTORSChoose two restriction enzymes, then answer questions. You can use EcoRI and HindIII if you must. What restriction enzymes did you use?Do you have to use the same enzymes for the cellular DNA andthe plasmid DNA? Explain.If DNA molecules are cut with the samerestriction enzyme, both will produce fragments withthe same complementary sticky ends, making it possiblefor DNA chimeras to form.What genes are on your recombinant plasmid?What agar plates would you choose for your control plates andyour experimental plates? Why?After transforming cells with the recombinant plasmid andplating on selective media with antibiotics, how will you identifypositive clones?Is it possible to find cells that did not take up the plasmid?Explain.Is it possible to find cells that took up the plasmid but don’thave the cellular gene incorporated into the plasmid?Explain.
01:41
You want to clone a human DNA sequence into a plasmid. This DNA fragment was cut out of the human genome with restriction enzyme SunI, whose restriction site is shown in the figure below.This figure also shows the multiple cloning region of your plasmid, with the potential restriction sites marked.
A. Which of these restriction enzymes could be used to cleave the plasmid for successful insertion of this human DNA fragment? Note that there could be more than one correct answer.
B. Briefly explain how you would go about cloning the fragment into the plasmid.
C. You successfully clone the human DNA into the plasmid and store it in the freezer. Several months later, your advisor asks you to use this recombinant plasmid to prepare a large quantity of the human insert sequence with as little plasmid sequence as possible. Can you do this with restriction enzymes? What enzymes would you choose?
01:54
You have been given a project to study the newly discovered aromatase-kinase H (AKH) 40 kDa protein. A colleague has isolated a 1400-bp Xho restriction fragment of human cDNA that molecular probes indicate contains the akh gene. Your assignment is to clone the gene to produce sufficient AKH for further study.Insert the restriction fragment containing the akh gene into the pGLO plasmid. Induce E. coli bacterial cells to take up the recombinant pGLO plasmid. Verify the presence of the pGLO plasmid carrying the akh gene in the bacteria by RFLP and PCR. Verify the expression of the akh gene in the bacteria by purifying the AKH protein from transformed bacterial cultures.
Transcript
So there’s a number of steps involved in cloning genes and the first is that both the plasmid and gene must be cut by the same enzyme. And restriction enzymes leave a sticky end which make it possible, if this is my gene, that’s my sticky end, and this is my plasmid with the same sticky end, this is the other side of the gene here, they act almost like a puzzle piece so that it’s very easy for these cloned pieces to fit very nicely into the plasmid. So number seven, the way that this fragment gets put into the plasmid is with something called ligase. So ligase is the enzyme that can make a covalent bond between the different fragments. So there’s a number. Now in question eight, you’re interpreting a set of restriction fragments that are used in DNA analysis for crime scenes and the victim is in lane one and we want to compare to the suspect in lane two. And in terms of comparing the bands, you want to look at thickness and size. So they should match up both of those traits and at this point, the only band that matches up exactly is labeled as band C because it is the same size and same thickness. So those are the only similarities between those two lanes. So there’s a number. After a bacteria has been…
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