Question

need help with this

Tm: Tm: 16. You wish to amplify the DNA fragments below and add cut sites for subsequent cloning. Design both primers with the restriction enzymes cut sites listed, and add two overhang bases to each primer. Label the extra bases and cut sites. Each primer should be 14 nucleotides in length. a. 5'-CTATGAGGTCCTGCGTTAGTGTTACC-3' Forward: 5'- 5' EcoRI, 3' BamHI, overhang bases Reverse: 5'- Annealing Temperature: b. 5'- CCTGGGTGTTACATGCCATTAGCGTT-3' Forward: 5'- Tm: 5' HindiII, 3' Sphl, overhang bases Reverse: 5'- Tm: Annealing Temperature: 17. Design the internal forward and reverse primers to mutate the amino acids shown. Primers are typically ~20 nucleotides in length, but only design these primers to be nine nucleotides. Label the mutated codon. a. 5'-TAAGGCTAGTAGACCGTATGCCGAATAC-3 Internal Forward: 5'- Mutate the threonine to cysteine (TGC) Internal Reverse: 5'-_ Annealing Temperature: b. 5'-CGCGTATGAGAGGATCTACTACGGATAG-3' Mutate the aspartate to tyrosine (TAT) Internal Reverse: 5'-- Tm: Annealing Temperature: Tm: Tm: Internal Forw ard:52 Tm:

Answer 1

CTATGAGGTCCTGCGTTAGTGTTACC

5’GCgaattcCTATGAGGTCCTGCGT3’    Tm 45.9 forward

5’GCggatccGGTAACACTAACGCAG3’ Tm 47.1 reverse

                             Annealing temperature (Ta) 41

CCTGGGTGTTACATGCCATTAGCGTT

5’ATaagcttCCTGGGTGTTACATGCC3’     Tm   49.5 forward

5CC’gcatgcA ACG CTA ATG GCATG3’   Tm   42.2 reverse

                                    Annealing temperature (Ta) 43

Mutageneic primers:

Threonine(ACC) to cystein(TGC)

TAAGGCTAGTAGTGCGTATGCCGAATAC

5'AGGCTAGTAGTGCGTATGCCGA3' Tm 56.7 Forward

5'TCG GCA TAC GCA CTA CTA GCC T 3' Tm 56.7 Reverse

                                      Annealing temperature (Ta) 52( 42-52 for gradient pcr)

Aspartate(GAT) to tyrosine(TAT)

CGCGTATGAGAGTATCTACTACGGATAG

5'GTATGAGAGTATCTACTACGGA3' Tm 51 forward

5'TCCGTAGTAGATACTCTCATA C3' Tm 51

                                        Annealing temperature (Ta) 46 ( 36-46for gradient pcr)