17. You wish to design a mutant of your protein (argval), and add a his tag. Des

Question

17. You wish to design a mutant of your protein (argval), and add a his tag. Design all four primers (entire primers, ~25-40 nucleotides each, written in 5’3’ direction) needed for PCR. For Tm’s include all overhangs/cut sites/tags, and use an online Tm calculator (search for one).

5’ end – add the 2 overhangs, a PstI cut site, a start codon, and a 6X his tag. Underline/label each component. 3’ end – add an XmaI cut site, and the 2 overhangs. Underline/label each component.
Internal primers – as before, but proper length with appropriate Tms. These do not contain cut sites or tags.

5’ATGTTGACTCCCTCACGGTGCCTCACGCTCACGGATGATAGTTTCCCGCTCTGCGATAG AGACCATAGGGCCGAGCGCGCCATAGAAAGGGCACTCCGCGCGCCATAAGCACTAGCCG GACTGATCGCGCAGCCACGTGGGCACGCGCGCCACATAATAGCCGAGAACAGCCCATGA 3’

A. External Forward: _____________________________________________________Tm:____

B. External Reverse: _____________________________________________________Tm:____

C. Internal Forward: ____________________________________ Tm:____

D. Internal Reverse: _____________________________________________________Tm:____

Explanation / Answer

  Pst1 recognition site    6X His tag                                                                                            Tm

A.5’AACTGCAGATGCATCATCATCATCATCATGTTTCCCGCTCTGCGATAGACCCGGG3’               88.82

B. 3’TTGACGTCTACGTAGTAGTAGTAGTAGTACATGGGCTGTTCTCGGCTATGGGCCC5’ 80.49

      Over hangs Start codon Met XmaI cut site

C. CCATAGAAAGGGCACTCCGC 60.82

D. CATGGGCTGTTCTCGGCTATT                                                                                             60.48

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