2. Creating an RNAi feeding vector

4. How does ligation into L4440 work?

5. Creating an RNAi feeding vector Isolate DNA from bacteria Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain Taq preferentially adds an extra “A” to the 3’ end of PCR products

6. Creating an RNAi feeding vector A A Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain Taq preferentially adds an extra “A” to the 3’ end of PCR products

7. Creating an RNAi feeding vector Taq will add a 3’ T to blunt-cut L4440 vector if there is only dTTP in the reaction buffer Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain L4440

8. Creating an RNAi feeding vector T T Taq will add a 3’ T to blunt-cut L4440 vector if there is only dTTP in the reaction buffer Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain L4440

9. Creating an RNAi feeding vector A A T T Ligate PCR product into T-tailed vector T T A A Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain L4440

10. Creating an RNAi feeding vector A A T T Ligate PCR product into T-tailed vector T T A A Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain Taq preferentially adds an extra “A” to the 3’ end of PCR products L4440

11. Creating an RNAi feeding vector A A T T Ligate PCR product into T-tailed vector T T A A Cut DNA with restriction enzymes Check DNA on gel Transform into the RNAi feeding strain Taq preferentially adds an extra “A” to the 3’ end of PCR products L4440

12. This is a very efficient and useful process: Works for any PCR product The extra T on the vector inhibits vector self-ligation

13. How does the vector induce RNAi? Allows E. coli to produces double-stranded RNA Requires a specific strain of E. coli

14. The RNAi feeding vector has two T7 RNA polymerase promoters T7 RNA polymerase is a viral polymerase with its own promoter sequence This allows transcription of the two strands of RNA from the insert between the promoters

15. T7 RNA polymerase E. coli strain HT115(DE3) has a modified lac promoter controlling the transcription of T7 RNA polymerase

16. Regulation of Lac Operon transcription IPTG also induces

17. When IPTG is added T7 RNA polymerase is expressed T7 RNA polymerase