5’ - TCAGATAAAAAACTGGCACGCAATCTGCAATTAGCAAGACATCTTTTTAGAACACGCTGAATAAATTGAGGTTGC

Question

5’ -
TCAGATAAAAAACTGGCACGCAATCTGCAATTAGCAAGACATCTTTTTAGAACACGCTGAATAAATTGAGGTTGCTATGTCTATTGTGGTGAAAAATAACATTCATTGGGTTGGTCAACGTGACTGGGAAGTGCGTGATTTTCACGGCACGGAATATAAAACGCTGCGCGGCAGCAGCTACAATAGCTACCTCATCCGCGAAGAAAAAAACGTGCTGATCGACACCGTCGACCATAAATTCAGCCGCGAATTTGTGCAGAACCTGCGTAATGAAATCGATCTGGCGGATATCGATTACATCGTGATTAACCATGCAGAAGAGGACCACGCTGGGGCGCTGACCGAACTGATGGCACAAATTCCCGATACGCCGATCTACTGTACAGCCAACGCTATCGACTCGATAAATGGTCATCACCATCATCCGGAGTGGAATTTTAATGTGGTGAAAACTGGCGACACGCTGGATATCGGCAACGGCAAACAGCTCATTTTTGTCGAAACACCAATGCTGCACTGGCCGGACAGCATGATGACTTACCTGACAGGCGACGCGGTGCTGTTCAGTAACGATGCTTTCGGTCAACACTACTGCGACGAGCATCTGTTCAACGATGAAGTGGATCAGACGGAGCTTTTCGAGCAGTGCCAGCGTTACTACGCCAATATCCTGACGCCGTTCAGCCGCCTGGTAACACCGAAAATTACCGAGATCCTGGGCTTTAACTTACCAGTCGATATGATAGCCACTTCCCACGGCGTGGTATGGCGCGATAACCCGACGCAAATTGTCGAGCTGTACCTGAAATGGGCGGCTGATTATCAGGAAGACAGAATCACCATTTTCTACGACACCATGTCGAATAACACCCGCATGATGGCTGACGCTATCGCCCAGGGGATTGCGGAAACCGACCCACGCGTGGCGGTGAAAATTTTCAACGTCGCCCGAAGCGATAAAAACGAAATCCTGACTAATGTCTTCCGCTCAAAAGGCGTGCTGGTCGGCACTTCGACGATGAATAACGTGATGATGCCGAAAATCGCCGGGCTGGTGGAGGAGATGACTGGTTTACGCTTCCGTAACAAACGCGCCAGTGCTTTCGGCTCTCACGGCTGGAGCGGCGGTGCGGTGGATCGTCTTTCCACGCGCCTGCAGGATGCGGGTTTCGAAATGTCGCTTAGCCTGAAAGCGAAATGGCGACCAGACCAGGACGCTCTGAAGTTATGCCGTGAACACGGTCGCGAAATCGCCCGTCAGTGGGCGCTCGCGCCGCTGCCGCAGAGCACGGTGAATACGGTAGTTAAAGAAGAAACCTCTGCCACCACGACGGCTGACCTCGGCCCACGGATGCAGTGCAGCGTCTGCCAGTGGATTTACGATCCGGCAAAAGGCGAGCCAATGCAGGACGTTGCGCCAGGAACGCCGTGGAGTGAAGTCCCGGATAACTTCCTCTGCCCGGAATGCTCCCTCGGCAAAGACGTCTTTGAAGAACTGGCATCGGAGGCAAAATGAGTAACGGCATTGTGATCATCGGTTCGGGCTTCGCCGCCCGCCAACTGGTGAAAAATATTCGCAAACAGGACGCCACTATTCCATT-3’

You wish to perform Quikchange mutagenesis on the gene of interest If you correctly translated the DNA sequence, the 194th amino acid encoded should be a tyrosine when you include the start methionine in your count. Design a primer that would allow you to convert tyrosine 194 to phenylalanine Y194F substitution). In addition to changing the amino acid identity, your primer should include at least 12-15 nucleotides on either side of the introduced changes, would ideally start and end with either a C or G, be between 25 and 45 nucleotides long and have a Tm of at least 78 oC. (Tm 81.5 0.41(%GC) (675 mismatch: and N primer length in nucleotides) a) What is the sequence of the forward primer you propose for this experiment? b) What is the Tm of the primer you designed? List the website or method you used for calculating the Tm since the calculated value depends on the method c) What is the sequence of the reverse primer you would employ to perform this Quikchange experiment? Remember to write your primers in the 5 3' direction)

Explanation / Answer

Forward Primer - 5’ CTGTCCCTGATGCTGAAGGCAGAGCAGAATGACTTC 3’

Tm of Primer (0.5 µM) is 69.4oC

Reverse Primer - 5’ GAAGTCATTCTGCTCTGCCTTCAGCATCAGGGACAG 3’

You can calculate Tm using following websites

https://www.thermofisher.com

http://www6.appliedbiosystems.com

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