Given the following restriction map of a vector, pET100D-TOPO, with the inserted

Question

Given the following restriction map of a vector, pET100D-TOPO, with the inserted gene PfIspC, what restriction enzyme would you choose to determine if the inserted gene is oriented correctly in the vector? What size fragments would you expect to see using the restriction enzyme you chose? What size fragments would you see if your gene were oriented backwards in the vector? (The PCR insert for PfIspC starts at bp 407and ends at bp 1650).

Created with SnapGene SgrAI (148 (6908) Pfol T7 promoter act operato NdeI (295) Nhel (333) Bmti (337) 7000 Scal (437) Psil (643) oter RBs Psil (1162) PshAI (5635 (5408) EagI SacI (1660) T7 terminator tet promoter) Acul (2321) ori 4000 Scal (2506) Pvul (2618) PstI (2745) (4229) Pfol (3992) BspQI (3875) Pcil Acul (3333) AlwNI (3466 PfispC Nterm in pET100DTOPO 7009 bp

Explanation / Answer

The enzymes I would choose will be ScaI and Sac I(1660). The insert size is 1243 bp. If the insert is orieted correctly as shown in figure, the fragments would be 846 bp,1223 bp and 4940bp.

If the insert is oriented backwards then one release would be smaller 40bp release, one 1223 bp and 5746 bp.

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