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Task #3: Design primers agagtctcct cagacgccga gatgctggtc atggcgcccc gaaccgtcct cctgctgctc 61 tcggcggccc tggccctgac cgagacctgg gccggtgagt gcgggtcggg agggaaatgg 121 cctctgccgg gaggagcgag gggaccgcag gcgggggcgc atgacctcag gagccgcgcc 181 gggaggaggg tcgggcgggt ctcagcccct cctcaccccc aggctcccac tccatgtggt 241 atttctacac ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc tcagtgggct 301 acgtggacga cacccagttc gtgaggttcg acagcgacgc cgcgagtccg agagaggagc 361 cgcgggcgcc gtggatagag caggaggggc cggagtattg ggaccggaac acacagatct 421 acaaggccca ggcacagact gaccgagaga gcctgcggaa cctgcgcttc tactacaacc 481 agagcgaggc cgttgcgtga ccccggcccg gggcgcaggt cacgactccc catcccccac 541 gtacggcccg ggtcgccccg agtctccggg tccgagatcc gcctccctga ggccgcggga a) First you'll need to design primers to PCR-amplify amino acids 45-56. i) Record the sequences of the forward and reverse primers, in the 5' to 3' direction. Each primer must be 8 nucleotides long (note that normally primers are much longer than this). Show intermediate steps in your primer design process and/or explain how you determined the primer sequences. ii) Using the method described in the pre-tutorial material, calculate the Tm for each primer (in °C). b) Next, you'll need to incorporate restriction sites into your primers. Shown here is the expression vector you want to clone the C-terminal domain into ("KanR2” is the antibiotic resistance gene): KanR2 pBR322 origin f1 origin T7 terminator Xhol (159) Eagl (167) NotI (167) HindIII (174) Sall (180) PET-28b(+) 5368 bp (Novagen) Sacl (191) EcoRI (193) Multiple Cloning Site (MCS) BamHI (199) Nhel (231) Ndel (238) Ncol (296) T7 promoter lacl i) Select a restriction enzyme (one that will generate sticky ends) that you could clone your fragment into, and indicate the sequence of its restriction site (e.g. in the format: Pstl, CTGCA/G). ii) Add the restriction enzyme site for the restriction enzyme you selected in (i) to each primer and re-write your primer sequences.

Task #3: Design primers agagtctcct cagacgccga gatgctggtc atggcgcccc gaaccgtcct cctgctgctc 61 tcggcggccc tggccctgac cgagacctgg gccggtgagt gcgggtcggg agggaaatgg 121 cctctgccgg gaggagcgag gggaccgcag gcgggggcgc atgacctcag gagccgcgcc 181 gggaggaggg tcgggcgggt ctcagcccct cctcaccccc aggctcccac tccatgtggt 241 atttctacac ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc tcagtgggct 301 acgtggacga cacccagttc gtgaggttcg acagcgacgc cgcgagtccg agagaggagc 361 cgcgggcgcc gtggatagag caggaggggc cggagtattg ggaccggaac acacagatct 421 acaaggccca ggcacagact gaccgagaga gcctgcggaa cctgcgcttc tactacaacc 481 agagcgaggc cgttgcgtga ccccggcccg gggcgcaggt cacgactccc catcccccac 541 gtacggcccg ggtcgccccg agtctccggg tccgagatcc gcctccctga ggccgcggga a) First you'll need to design primers to PCR-amplify amino acids 45-56. i) Record the sequences of the forward and reverse primers, in the 5' to 3' direction. Each primer must be 8 nucleotides long (note that normally primers are much longer than this). Show intermediate steps in your primer design process and/or explain how you determined the primer sequences. ii) Using the method described in the pre-tutorial material, calculate the Tm for each primer (in °C). b) Next, you'll need to incorporate restriction sites into your primers. Shown here is the expression vector you want to clone the C-terminal domain into ("KanR2” is the antibiotic resistance gene): KanR2 pBR322 origin f1 origin T7 terminator Xhol (159) Eagl (167) NotI (167) HindIII (174) Sall (180) PET-28b(+) 5368 bp (Novagen) Sacl (191) EcoRI (193) Multiple Cloning Site (MCS) BamHI (199) Nhel (231) Ndel (238) Ncol (296) T7 promoter lacl i) Select a restriction enzyme (one that will generate sticky ends) that you could clone your fragment into, and indicate the sequence of its restriction site (e.g. in the format: Pstl, CTGCA/G). ii) Add the restriction enzyme site for the restriction enzyme you selected in (i) to each primer and re-write your primer sequences.

Human Anatomy & Physiology (11th Edition)
Human Anatomy & Physiology (11th Edition)
11th Edition
ISBN:9780134580999
Author:Elaine N. Marieb, Katja N. Hoehn
Publisher:Elaine N. Marieb, Katja N. Hoehn
Chapter1: The Human Body: An Orientation
Section: Chapter Questions
Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...
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Task #3: Design primers agagtctcct cagacgccga gatgctggtc atggcgcccc gaaccgtcct cctgctgctc 61 tcggcggccc tggccctgac cgagacctgg gccggtgagt gcgggtcggg agggaaatgg 121 cctctgccgg gaggagcgag gggaccgcag gcgggggcgc atgacctcag gagccgcgcc 181 gggaggaggg tcgggcgggt ctcagcccct cctcaccccc aggctcccac tccatgtggt 241 atttctacac ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc tcagtgggct 301 acgtggacga cacccagttc gtgaggttcg acagcgacgc cgcgagtccg agagaggagc 361 cgcgggcgcc gtggatagag caggaggggc cggagtattg ggaccggaac acacagatct 421 acaaggccca ggcacagact gaccgagaga gcctgcggaa cctgcgcttc tactacaacc 481 agagcgaggc cgttgcgtga ccccggcccg gggcgcaggt cacgactccc catcccccac 541 gtacggcccg ggtcgccccg agtctccggg tccgagatcc gcctccctga ggccgcggga a) First you'll need to design primers to PCR-amplify amino acids 45-56. i) Record the sequences of the forward and reverse primers, in the 5' to 3' direction. Each primer must be 8 nucleotides long (note that normally primers are much longer than this). Show intermediate steps in your primer design process and/or explain how you determined the primer sequences. ii) Using the method described in the pre-tutorial material, calculate the Tm for each primer (in °C). b) Next, you'll need to incorporate restriction sites into your primers. Shown here is the expression vector you want to clone the C-terminal domain into ("KanR2” is the antibiotic resistance gene): KanR2 pBR322 origin f1 origin T7 terminator Xhol (159) Eagl (167) NotI (167) HindIII (174) Sall (180) PET-28b(+) 5368 bp (Novagen) Sacl (191) EcoRI (193) Multiple Cloning Site (MCS) BamHI (199) Nhel (231) Ndel (238) Ncol (296) T7 promoter lacl i) Select a restriction enzyme (one that will generate sticky ends) that you could clone your fragment into, and indicate the sequence of its restriction site (e.g. in the format: Pstl, CTGCA/G). ii) Add the restriction enzyme site for the restriction enzyme you selected in (i) to each primer and re-write your primer sequences.
Transcribed Image Text:Task #3: Design primers agagtctcct cagacgccga gatgctggtc atggcgcccc gaaccgtcct cctgctgctc 61 tcggcggccc tggccctgac cgagacctgg gccggtgagt gcgggtcggg agggaaatgg 121 cctctgccgg gaggagcgag gggaccgcag gcgggggcgc atgacctcag gagccgcgcc 181 gggaggaggg tcgggcgggt ctcagcccct cctcaccccc aggctcccac tccatgtggt 241 atttctacac ctccgtgtcc cggcccggcc gcggggagcc ccgcttcatc tcagtgggct 301 acgtggacga cacccagttc gtgaggttcg acagcgacgc cgcgagtccg agagaggagc 361 cgcgggcgcc gtggatagag caggaggggc cggagtattg ggaccggaac acacagatct 421 acaaggccca ggcacagact gaccgagaga gcctgcggaa cctgcgcttc tactacaacc 481 agagcgaggc cgttgcgtga ccccggcccg gggcgcaggt cacgactccc catcccccac 541 gtacggcccg ggtcgccccg agtctccggg tccgagatcc gcctccctga ggccgcggga a) First you'll need to design primers to PCR-amplify amino acids 45-56. i) Record the sequences of the forward and reverse primers, in the 5' to 3' direction. Each primer must be 8 nucleotides long (note that normally primers are much longer than this). Show intermediate steps in your primer design process and/or explain how you determined the primer sequences. ii) Using the method described in the pre-tutorial material, calculate the Tm for each primer (in °C). b) Next, you'll need to incorporate restriction sites into your primers. Shown here is the expression vector you want to clone the C-terminal domain into ("KanR2” is the antibiotic resistance gene): KanR2 pBR322 origin f1 origin T7 terminator Xhol (159) Eagl (167) NotI (167) HindIII (174) Sall (180) PET-28b(+) 5368 bp (Novagen) Sacl (191) EcoRI (193) Multiple Cloning Site (MCS) BamHI (199) Nhel (231) Ndel (238) Ncol (296) T7 promoter lacl i) Select a restriction enzyme (one that will generate sticky ends) that you could clone your fragment into, and indicate the sequence of its restriction site (e.g. in the format: Pstl, CTGCA/G). ii) Add the restriction enzyme site for the restriction enzyme you selected in (i) to each primer and re-write your primer sequences.
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