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. Author manuscript; available in PMC: 2014 Nov 5.
Published in final edited form as: Curr Protoc Microbiol. 2013 Nov 5;31:15K.1.1–15K.1.45. doi: 10.1002/9780471729259.mc15k01s31

Figure 3. Cloning Strategy for inserting norovirus ORF2 into pVR21.

Figure 3

Step 1) Using plasmid DNA from Basic Protocol 3, an Asc1 site and a VEE linker sequence are added to ORF2 and amplified through insert PCR. Using pVR21, an overlapping region of VEE is amplified through VEE arm PCR. Step 2) Using Step 1 PCR products, a single overlap PCR product is generated that contains both the VEE arm and norovirus capsid gene. Step 3) The overlap PCR product is cloned into a TOPO TA plasmid. Step 4) Both the plasmid from Step 3 and pVR21 are cut using Apa1 and Asc1 enzymes. Step 5) The norovirus capsid gene is ligated into pVR21.