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A plasmid having a restriction site for Hind III enzyme within a kanamycin resistance gene was cleaved with Hind III and was re-ligated. It was used to transform E.coli (Escherichia coli) K12 cells. From the culture plate, kanamycin resistant colonies were selected and electrophoresis of the plasmid DNA from these colonies was carried out. Most of the colonies with plasmid produce a single band, which migrated at the same speed as the original intact plasmid. A slow band in agarose gel electrophoresis was obtained as a product of ligation.
Introduction:
Agarose gel electrophoresis is standard lab procedure for a DNA (deoxyribonucleic acid) separation on the basis of size (length in base pair). Agarose gel electrophoresis uses an electrical field to mobilize negatively charged DNA molecule in an agarose gel matrix toward the positively charged electrode. A DNA molecule, when digested with restriction enzymes (tools of recombinant DNA technology or RDT), produces the bands of different sizes when subjected to electrophoresis. This technique is used for diagnostic purposes to determine the genetic defects.
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Essentials of Genetics
- The gene atg-12 codes for a protein associated with abnormal rates of cell destruction and recycling of cell contents. Scientists studying bacterial plasmids devised an experiment using recombinant DNA techniques to remove a section of DNA (gene atg-12) of a bacterial plasmid (pOKE103) and create a new plasmid (pOKE104) that did not contain the gene atg-12. The new plasmid was then incorporated within the DNA of the fungus Neurospora crassa in cellular studies. Which statement explains the expected heredity of fungi that incorporate pOKE104? A - Fungi that incorporate pOKE104 will produce the protein from gene atg-12 and have increased rates of malignant tumors. B - Fungi that incorporate pOKE104 will not produce the protein from gene atg-12 but will have a normal appearance. C - The prokaryotic DNA will remain separate within the fungal cells, produce the protein from gene atg-12, and have increased rates of malignant tumors. D - The prokaryotic DNA will remain separate within the…arrow_forwardA plasmid that is both ampicillin and tetracyclineresistant is cleaved with PstI, which cleaves within theampicillin resistance gene. The cut plasmid is ligated withPstI-digested Drosophila DNA to prepare a genomic library,and the mixture is used to transform E. coli K12. Question: If recombinant cells were plated on medium containingampicillin or tetracycline and medium withboth antibiotics, on which plates would you expectto see growth of bacteria containing plasmids withDrosophila DNA inserts?arrow_forwardWhen cloning a foreign DNA fragment into a plasmid, it is often useful to insert the fragment at a site that interrupts a selectable marker(such as the tetracycline-resistance gene of pBR322). The loss of function of the interrupted gene can be used to identify clones containing recombinant plasmids with foreign DNA. With a yeast artificial chromosome (YAC) vector, it is not necessary to do this; the researcher can still distinguish vectors that incorporate large foreign DNA fragments from those that do not. How are these recombinant vectors identified?arrow_forward
- Find a plasmid map for pET11a and create a basic procedure for cloning a gene into this vector. Which selection method and substance would you use for this plasmid after transformation?arrow_forwardKnowing that you are using HindIII and EcoRI to cut your plasmids, and that those two enzymes cut within the MCS, use the map of pUC19 provided below to compute: What will be the sizes of the 2 restriction fragments if NO insert is present in pUC19? What will be the sizes of the 2 restriction fragments if the approximately 317 bp RT-PCR product (insert from WT satC dimer) was ligated successfully into the SmaI site? What will be the sizes of the 2 restriction fragments if TWO approximately 317 bp RT-PCR products (2 ligated inserts from WT satC dimer) were ligated into the SmaI site?arrow_forwardWhen using a conventional plasmid cloning vector containing a b-galactosidase gene, it is possible to perform a "blue-white screen" to determine which bacteria have taken up a plasmid into which a DNA fragment as been inserted, as opposed to those that have taken up just reclosed plasmid vector, by growing the transformed cells on nutrient agar plates containing the artificial b-gal substrate X-gal. Will bacteria that have taken up a plasmid into which a DNA fragment has been inserted form a blue colony or a white colony when grown on this medium? Briefly explain why these bacteria would form a colony of the color you chose.arrow_forward
- When a 5-kb circular plasmid is digested with a restriction enzyme that has three recognition sites on the plasmid, how many bands can be visualized on an agarose gel? a)1 b)2 c)5 d)3arrow_forwardYou have a recombinant plasmid containing a vector and a segment of foreign DNA, both equal sizes. Draw a picture of this recombinant plasmid labeling foreign and vector regions. Where the foreign DNA meets the vector, there is a cut site for restriction enzyme ABC1. When the recombinant plasmid is cut by ABC1, how many fragments do you expect to be produced? Identify these fragments.arrow_forwardThis plasmid was digested using different restriction enzymes whose sites have been mapped. The plasmid is 7896 base pairs long. This is a long question so u can count this as two or even three but please answer the question? Determine the size (base pairs) and number of fragments that would be produced if the plasmid was digested with the following enzymes: a) EcoRI b) BamHI c) HindIII d) EcoRI and HindIII e)EcoRI, HindIII, and BamHI *Hint- this is actually an EASY question, since the restriction map is already drawn for you!arrow_forward
- One would be correct in assuming that the restriction sites present in the multiplecloning site of the pUC18 plasmid willarrow_forwardA PCR reaction was performed to amplify the XULA3 gene, which is bp 882-5,364 on a plasmid that is 11,719 bp. After the PCR, the product was digested with XhoI. There are XhoI sites on the plasmid at bp 1,434, 4,655, and 7,368. Calculate the size(s) that would result when the product is digested with XhoI. Then enter the size of the largest fragment (in bp).arrow_forwardThe total size of the plasmid is 2686 bp. There is a PstI recognition site at position 439, HindIII recognition site at position 447, and ScaI recognition site at 2179. If restriction enzymes ScaI and HindIII are used to cut this plasmid, what would be produced? a. 2 fragments: 954bp and 1732bp b. none of the choices apply c. 1 linear fragment of 2686bp d. 3 fragments: 447bp, 507bp and 1732bparrow_forward
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