Data Sheet: Restriction Analysis of Plasmid DNA 201 261 311 Reverse priming site T3 priming site CACACAGGAA ACACCIAIGA C

[answerhappygod]

Data Sheet Restriction Analysis Of Plasmid Dna 201 261 311 Reverse Priming Site T3 Priming Site Cacacaggaa Acacciaiga C 1 (115.45 KiB) Viewed 26 times

Data Sheet: Restriction Analysis of Plasmid DNA 201 261 311 Reverse priming site T3 priming site CACACAGGAA ACACCIAIGA CCATOADTAC GCCAAGOTCA GAATTAACCO TCACTAAAGG in GESTOTOOP ICTOGATACT GGLADIAATO SOGTICOACT Sce STOR GASTACTO -Mon LacZainitation code Predd 12000G CIIAAGOGOO M13 Forward (-20 ming to — COGOTAAAIT CAATTOOCCO TATAGIGAGI COIATTACAA 11CASTGGOO GUCATITAS GEOGAITTAA SITAAGOGGG AZAZCACICA GCATAAIGII AAGIGACOGG CASCAAAATO Lacza-coda P You can assume Spel, Pstl, Pmel, EcoRI, and Notl only cut the PCR4-TOPO vector sequence at the locations shown here. However, you would need the sequence of any PCR product insert to know if there are additional enzyme cut sites for a vector+insert plasmid product. P ATALIALA AGOTTHAY ACGRATITnan yh A COLCCARAII IGOTIAAGOG SOHA arming sit Size of vector: orl PIAC PCR 4-TOPOⓇ 3956 bp ગdurd PCR PCR Product Molecular Module MAN LAVALCA naluno Size of insert: Kanamycin EcoR 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? Not dit 2.) Which enzyme option will best help us with this verification step, regardless of the PCR product insert used: Spel, Pstl, Pmel, EcoRI, or Notl? Why? 6.) What size (kb) would the resulting fragment(s) be? Which primer set is your group using for the practice digest prediction example below? 3.) If your WT PCR product from this primer set was inserted into the pCR4-TOPO vector, what is the total size of the plasmid? Estimate size in kilobase pairs (kb), round to the closest decimal point. kb 4.) How many times will Notl cut this TOPO + WT product plasmid? (Assume for this practice example that Notl does not cut your WT product insert) 5.) How many fragments would result from a Notl digest of this TOPO + WT product plasmid?