Molecular Module Data Sheet: Restriction Analysis of Plasmid DNA e Leeza initiation codon M13 Reverse priming pie 73 gaming site 201 CACACAGGAA ACAGOTATGA CCATGATTAG CCCARGCICA GAATTAACCE TOACTAAAGG GTGTGTCCTT TOTCOATACT GOTACTAATG COOTTOGAGT CITAATTOGG AGTGATTTCC 1 Pmal Econ Nor 261 GACTAGTOCT GCAGGTTTAA ACCAATTCGS CCIT PCR AJAGGOC CAATICOCGG CTGATCAGGA CGTCCAAATT TOCTTAAGCG GGAA Product PICCCG CITAAGCOCC 17 priming site M13 Forward (-20) priming ate 311 CCGCTAAATT CAATTCOCCO CCCCCC TATAGTGAGT CGTATTACAA TTCACTGGCC GTCOTTITAC GOCGATTTAA GTTAAGCGGG ATATCACTCA GCATAATGTT AAGTGACCGG CACCAAAATG You can assume Spel, Pstl, P IBC LacZa.cedB Pmel, EcoRI, and Notl only cut the pCR4-TOPO vector sequence at the locations PUC ori shown here. However, you would need the sequence of PCR 4-TOPO any PCR product insert to 3956 bp know if there are additional Kanamycin enzyme cut sites for a vector+insert plasmid product. Ampicillin 1.) Why perform a restriction digest after cloning your gene of interest fragments into the TOPO vector? What do we want to verify before proceeding to the final sequencing step? confirms presence of gine of interest 2.) Which enzyme option will best help us with this verification step, regardless of the PCR product insert used: Spel, Pstl, Pmel, EcoRI, or Noti? Why? Which primer set is your group using for the practice digest prediction example below