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Chrome File Edit View History Bookmarks People Window Help 4) us. 100% Gr Mon Mar 12 12:30 AM Q E Primer design question 1 Secure https:// portal.utoronto.ca/bbcswebdav/pid-6620776-dt-content-rid-43684942-2/courses/Winter-2018-BIO3 14H5-S-LECO101/Primer… a : Apps Maria Robinson qu Sports TV 1-watc USM Management l Ran Fred Perry Footwea V sports-Live Video CE is,byu.edu/courses e Other Bookmarks l : Watch Arsenal vs S Primer design question 1 2018.pdf Problem based on material authored by Nick Harner University of Exeter, UK Designing reverse primer of a primer pair. Assume this is the sequence to which you would like your primer to bind ATCGAGTCAGGGTCCAA You would also like to add an Ndel restriction site to the primer, so that you can cut the PCR product. Nde1 will not cut well near the end of a DNA fragment, so you need to add an additional 3 bases. For your interest, here is an NEB guide to the cutting efficiency of different restriction enzymes a the ends of DNA: https://www.neb.com/tools-and-resources usage- What is the sequence of the primer? Always write this 5'-3 Examine your primer what problems do you foresee with it? 0 Can you come up with alternative methods to clone your PCR product?

Explanation / Answer

Sequence: 5' ATCGAGTCAGGGTCCAA
Nde1 restriction site: 5'CATATG

combined sequence : 5' CATATGATCGAGTCAGGGTCCAA

lets add 3 additional bases: 5' ACCCATATGATCGAGTCAGGGTCCAA 3'

primer 1 : 5' ACCCATATGATCGAGTCAGGGTCCAA 3'
primer 2 : 3' TTGGACCCTGACTCGATCATATGGGT 5'

This primer has many repeatitive bases, which might result in hairpin loops. The primer can also form primer dimer.

We can add any other restriction enzyme sequence which can result in less of primer dimers.We can use homologous recombination to exchange a small region of DNA with the restriction sites.

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