The restricition EcoRI cleaves double-stranded DNA at the sequence 5'-GAATTC-3' the restriction enzyme Hindll cleaves at the sequence 5'-AAGCTT-3'. A 20 kb circular plasmid containing OFP gene is digested with each enzyme individually and then in combination, and the resulting fragment sizes are determined using electrophoresis Restriction Enzyme(s) ECORI alone Fragment length 6 kb ,14 kb 7 kb , 13 kb 2kb, 4kb, 5 kb, 9kb Hindlll alone EcoRI and Hindll Construct the circular molecule and indicate the relative positions of the EcoRI and Hindlll restriction sites

The restricition EcoRI cleaves double-stranded DNA at the sequence 5'-GAATTC-3' the restriction enzyme Hindll cleaves at the sequence 5'-AAGCTT-3'. A 20 kb circular plasmid containing OFP gene is digested with each enzyme individually and then in combination, and the resulting fragment sizes are determined using electrophoresis Restriction Enzyme(s) ECORI alone Fragment length 6 kb ,14 kb 7 kb , 13 kb 2kb, 4kb, 5 kb, 9kb Hindlll alone EcoRI and Hindll Construct the circular molecule and indicate the relative positions of the EcoRI and Hindlll restriction sites

Name: The restricition EcoRI cleaves double-stranded DNA at the sequence 5'
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Description: The restricition EcoRI cleaves double-stranded DNA at the sequence 5'-GAATTC-3' the restrictionenzyme Hindll cleaves at the sequence 5'-AAGCTT-3'. A 20 kb circular plasmid containing OFPgene is digested with each enzyme individually and then in comb

Biochemistry

6th Edition

ISBN:9781305577206

Author:Reginald H. Garrett, Charles M. Grisham

Publisher:Reginald H. Garrett, Charles M. Grisham

Chapter28: Dna Metabolism: Replication, Recombination, And Repair

Section: Chapter Questions

Problem 10P: Homologous Recombination, Heteroduplex DNA, and Mismatch Repair Homologous recombination in E. coli...

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Homologous Recombination, Heteroduplex DNA, and Mismatch Repair Homologous recombination in E. coli leads to the formation of regions of heteroduplex DNA. By definition, such regions contain mismatched bases. Why doesnâ€™t the mismatch repair system of E. coli eliminate these mismatches?
Heteroduplex DNA Formation in Recombination From the information in Figures 28.17 and 28.18, diagram the recombinational event leading to the formation of a heteroduplex DNA region within a bacteriophage chromosome.
The recognition sequence for the restriction enzyme Sau3AIis GATC; in the recognition sequence forthe enzyme BamHI—GGATCC—the four internal bases areidentical to the Sau3AI sequence. The single-stranded endsproduced by the two enzymes are identical. Suppose youhave a cloning vector that contains a BamHI recognitionsequence and you also have foreign DNA that was cut withSau3AI. Can this DNA be ligated into the BamHI site of the vector,and if so, why?
U have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. PscI & GsuI ______________________________________________________________ ScaI, PdmI & BsaXI ______________________________________________________________ ScaI, SspI & EheI ______________________________________________________________
1. You have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions. a. PscI & GsuI b. ScaI, PdmI & BsaXI c. ScaI, SspI & EheI 2. You have determined that the amplicon you want to clone has enzyme restriction sites for HindIII and EcoRI. After investigation you have seen that the pUC18/19 plasmid also have these enzyme restriction sites in its multiple cloning site (MCS on map above). After enzyme digestion your amplicon is 854 bp long. a. What length will the recombinant plasmid be after you have inserted your amplicon? Show your calculation. b. In the amplicon insert you have an enzyme restriction site for NdeI at 500 bp. If you digest the recombinant plasmid with this enzyme what length will the fragments be?
a)Dr. Thisisaneasyexam decides to amplify a gene from a plasmid using PCR. She starts out with 6.6 x 10-14g of a 10 kb template in a 100 µl reaction. Assuming that the molecular weight of the average base pair is 660 Daltons calculate the number of molecules of the template. b)Given that the concentration of each primer (20 base pairs each) is 0.1µM in this same reaction volume (100 µl) calculate the number of molecules of the primer present
1. You have the plasmid pUC18/19, which is a circular plasmid that consists of 2686 bp. What would the number of and length of the fragments be if you cut the plasmid with the following restriction enzymes or combination of enzymes? Give a schematic representation of the digestions.a. PscI & GsuIb. ScaI, PdmI & BsaXI c.ScaI, SspI & EheI
RNA polymeraze binds 100x quicker to the promotor when the promotor is in a 8kb plasmid than to the same promotor on a 200bp linear segment. Assuming that the incubation is performed with equal concentrations of the two types of DNA, explain this difference.
Do virtual restriction digests of 300ng pGLO plasmid using HinDIII, EcoRI, and XhoI separately, and a double digest of pGLO with HinDIII plus EcoRI. How many nanograms of DNA should we expect to be contained within the slowest bandfrom the HinDIII-digest of pGLO?
The restriction endonuclease PstI cuts DNA symmetrically on both strands at the sequence: CTGCAG GACGTC On the resulting DNA fragments are left 3’ overhanging ends of 4 nucleotides. PstI cleavesthe phosphodiester backbone with a-type specificity. In short-hand notation, showing theidentity of phosphates on the ends, draw the two fragments that result from PstI digestion ofthe following double-strand ed oligonucleotide: 5’-pAATTGCTACTGCAGAACCGG-3’ 3’-TTAACGATGACGTCTTGGCCp-5’
I. You wish to perform an electrophoretic resolution of your restriction enzyme–digested DNA. The sizes of the expected fragments range from 100 to 500 bp. You discover two agarose gels polymerizing on the bench. One is 0.5% agarose; the other is 2% agarose. Which one might you use to resolve your fragments? I. What does it mean to “denature” a protein and why is this important for SDS PAGE? II. Describe how you denatured the proteins you used for SDS-PAGE. III. Describe the roles of the primary Antibody and the secondary Antibody during an ELISA test. IV. After the addition of the secondary Antibody, how did you determine which wells contain the Antigen bound to primary Antibody bound to secondary Antibody?
Consider the following plasmid (size 8000 bp), with restriction sites at the positions indicated: (see image) a) This plasmid is digested with the enzymes listed below. Indicate how many fragments will begenerated in each case, and give the sizes of the fragments.PstIXhoICombination of PstI + XhoI + EcoRI (triple digest) b) Draw the banding pattern you would expect to observe if each of these digestions is loaded into a separate well of an agarose gel, and the fragments separated by electrophoresis. In the first well you load a DNA marker (M) containing fragments with sizes of 1000 bp, 2000 bp, 4000 bp and 8000 bp. c) This gel is transferred to a membrane in a Southern blot experiment, and hybridised to a radioactively labelled 200 bp probe, which anneals to the plasmid DNA at the position indicated on the diagram above. Draw the autoradiographic profile you would expect to observe for the membrane.