Concept explainers
To review:
The sequential steps for inserting a foreign DNA (deoxyribonucleic acid) into a plasmid or vector by arranging the below-mentioned steps in a correct order:
a. Transform host cells.
b. Select colonies for antibiotic resistance.
c. Select colonies that do not grow under ultraviolet light.
d. Digest vector and foreign DNA with a restriction enzyme.
e. Ligate the digested plasmid together with the foreign DNA.
Introduction:
Plasmids are small, extra-chromosomal DNA molecules. They are usually circular and are relatively smaller (2000–6000 base pairs). There are often multiple restriction sites present in a plasmid sequence. Thus, this makes it easier for the insertion of a new foreign DNA. In the given case, it is assumed that a plasmid vector contains genes for green fluorescent protein (GFP) as well as for ampicillin resistance. A restriction site is present on the gene for GFP only. A foreign DNA needs to be inserted into this plasmid, which in turn, is to be inserted into the bacteria.
Want to see the full answer?
Check out a sample textbook solutionChapter 18 Solutions
EBK LIFE: THE SCIENCE OF BIOLOGY
- Assume that a circular plasmid is 3200 base pairs in length and has restriction sites for HindIII restriction enzyme at the following locations: 400, 700, 1400, 2600. Give the expected sizes of the restriction fragments following complete digestion.arrow_forwardtrue or false if a restriction enzyme recognizes the restriction site, 5' AACGTT3', and the enzyme cuts between the second A and the C, this will produce a "sticky end," which is useful for cloning into a plasmid vector.arrow_forwardConsider a partial restriction digestion, in which genomic DNA is exposed to a small, limiting amount ofa restriction enzyme for a very short period of time.a. Would the resultant fragments be longer or shorteror the same size as those produced by a completedigestion?arrow_forward
- A. A plasmid is shown with the locations of various restriction enzyme sites labeled. If you cut the plasmid with Xhol and Xbal, which lane of the agarose gel represents the DNA fragments you would expect from the digestion? B. If you now decide to cut the plasmid with EcoRI, how many fragments will be produced and what will their sizes be? C. When running DNA samples on agarose gel, an electric field is applied. Towards which electrode will the DNA migrate and why?arrow_forwardYou are studying a new plasmid, and you digest the plasmid with three restriction enzymes: Eco RI (E), HindlII (H), and Xbal (X). You digest the plasmid DNA with each of the following combinations of enzymes and observe the results on an agarose gel. You are provided a partial plasmid map as shown below to the right. E+H E+X H+x Kb +4.3 +2.8 -+2.5 -2.0 - -1.8 +1.5 -1.0 12 F0.8 +0.5 a. What is the size of this plasmid in base pairs? b. What is the distance in base pairs between E1 and H? c. What is the distance in base pairs between E1 and X? d. What is the distance in base pairs between E2 and H? e. What is the distance in base pairs between E2 and X?arrow_forwardAssume that a plasmid is 4700 base pairs in length and has restriction sites for a given restriction enzyme at the following locations: 800, 1400, 2900, and 3600. List the fragments by size that are ! expected when the plasmid is fully digested the restriction enzyme.arrow_forward
- In relation to the use of restriction enzymes in recombinant DNA technology, answer the following: You have accidentally torn the labels off two tubes (tube A and tube B), each containing a different plasmid, now you do not know which plasmid is in which tube. Fortunately, you have restriction maps for both plasmids, shown in Figure below. You have the opportunity to test just one sample from one of your tubes. By utilizing agarose gel electrophoresis technique, which restriction enzyme OR combination of restriction enzymes would you use in this experiment to determine which plasmid is found in which tube?. (Hint: if you use Hind III restriction enzyme you are going to get ONE single fragment with a molecular size of → 0.5+0.3+0.2+0.4+1+1 = 3.4 kb).arrow_forwardPlease DESCRIBE, in outline form, the method you will use to select for bacterial cells that have taken up the pL311 plasmid, and to screen those cells for the presence of plasmids that are likely to contain a cloned gene. Be sure to mention the specific media you will use. In addition, please explain the rationale behind this specific selection and screening procedure. (Remember that you have available the following types of media: (i) media containing neither kanamycin nor X-gal, (ii) media containing BOTH kanamycin and X-gal, (iii) media containing tetracycline, and (iv) media containing ampicillin.arrow_forwardConsider a partial restriction digestion, in which genomic DNA is exposed to a small, limiting amount ofa restriction enzyme for a very short period of time.a. Would the resultant fragments be longer or shorteror the same size as those produced by a completedigestion?b. If you prepared genomic DNA from a tissue sample containing millions of cells, would the fragments produced by partial digestion of DNA fromall of these cells be the same or different?arrow_forward
- Please answer both Part a. The plasmid you used as a template in your positive control for your PCR is 3300 bp in size. Why is the PCR product only 650 bp long? b. You using PCR to check for the presence of the GFP gene, however the thermocyder (machine that through temperatures) has been misprogrammed and tge annealing temperature is set to 43c (low for your primers) Explain what you would expect to see on your gel and why?arrow_forwardA plasmid DNA and a linear DNA (both of the same size) have one site for a restriction endonuclease. When cut and separated on agarose gel electrophoresis, plasmid shows one DNA band while linear DNA shows two fragments. Explain.arrow_forwardA result of agarose gel electrophoresis of plasmid DNA is shown below. The direction of the electrophoresis is indicated by the arrow head. Lane 1 shows the plasmid DNA with no restriction enzyme digestion. Lane 2 shows the plasmid DNA which has been digested by restriction enzyme ECORI. Given the fact that there are only relaxed and supercoiled forms existing in this plasmid, please annotate the bands in Lane 1 on this gel with their supercoiling status (supercoiled or relaxed) and explain why. Then based on the information from Lane 1 and Lane 2, analyze the number of EcoRI cleavage sites on this plasmid. Lane 1 2arrow_forward
- Human Anatomy & Physiology (11th Edition)BiologyISBN:9780134580999Author:Elaine N. Marieb, Katja N. HoehnPublisher:PEARSONBiology 2eBiologyISBN:9781947172517Author:Matthew Douglas, Jung Choi, Mary Ann ClarkPublisher:OpenStaxAnatomy & PhysiologyBiologyISBN:9781259398629Author:McKinley, Michael P., O'loughlin, Valerie Dean, Bidle, Theresa StouterPublisher:Mcgraw Hill Education,
- Molecular Biology of the Cell (Sixth Edition)BiologyISBN:9780815344322Author:Bruce Alberts, Alexander D. Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter WalterPublisher:W. W. Norton & CompanyLaboratory Manual For Human Anatomy & PhysiologyBiologyISBN:9781260159363Author:Martin, Terry R., Prentice-craver, CynthiaPublisher:McGraw-Hill Publishing Co.Inquiry Into Life (16th Edition)BiologyISBN:9781260231700Author:Sylvia S. Mader, Michael WindelspechtPublisher:McGraw Hill Education