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Creating an RNAi feeding vector

Creating an RNAi feeding vector. How does ligation into L4440 work?. Creating an RNAi feeding vector. Taq preferentially adds an extra “A” to the 3’ end of PCR products. Isolate DNA from bacteria. Cut DNA with restriction enzymes. Check DNA on gel. Transform into the RNAi feeding strain.

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Creating an RNAi feeding vector

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  1. Creating an RNAi feeding vector

  2. How does ligation into L4440 work?

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

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

  5. Creating an RNAi feeding vector L4440 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

  6. Creating an RNAi feeding vector T T L4440 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

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

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

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

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

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

  12. 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

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

  14. IPTG also induces Regulation of Lac Operon transcription

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

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