Data Sheet: Restriction Analysis of Plasmid DNA 201 261 311 Laaton so You can assume Spel, Pstl, Pmel, EcoRI, and Notl o

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Data Sheet Restriction Analysis Of Plasmid Dna 201 261 311 Laaton So You Can Assume Spel Pstl Pmel Ecori And Notl O 1 (95.97 KiB) Viewed 15 times

Data Sheet: Restriction Analysis of Plasmid DNA 201 261 311 Laaton so 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. farm prog CACACASSAA ACASCSANGA SCATGATIAS OCASICA CAATTAADOO TOASTAANDO 3101010 13TOATACI OSTACAA13 COSTICAS CITAATIOSO ADIDAS CO Sco Pri Pry Ecomi Nati CASTAGIOCE SEADISTAN AGAATTOOS coo PCR CIGAICASSA COCCAAAIT TOCITAASES OAK Product TT sriming at H.W Plas ALADDOT SAANadan BOOOED DIIAA30000 M13 Ferwars 20 ming COGCAAATS CAATTedece TATAGOGAST COTATIACAA 11CAC10000 OECOUSETAS SOGATTAA SITAAGC003 ATASCACICA CATAATSTE AASTBASSSS CASCAAAA Lacza-ccde PCR 4-TOPOⓇ 3956 bp ગdurd Deng + Molecular Module E 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? Kanamycin 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. Size of insert: Size of vector: 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) 27 5.) How many fragments would result from a Notl digest of this TOPO + WT product plasmid? kb