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

Creating an RNAi feeding vector

AA

Taq preferentially adds an extra “A” to the 3’ end of PCR products

Creating an RNAi feeding vector

Taq will add a 3’ Tto blunt-cut L4440 vector if there is only dTTP in the reaction buffer

L4440

Creating an RNAi feeding vector

T T

Taq will add a 3’ Tto blunt-cut L4440 vector if there is only dTTP in the reaction buffer

L4440

Creating an RNAi feeding vector

AA

T T

Ligate PCR productinto T-tailed vector

T TA

A

L4440

L4440

Creating an RNAi feeding vector

AA

T T

Ligate PCR productinto T-tailed vector

T TA

A

Taq preferentially adds an extra “A” to the 3’ end of PCR products

L4440

L4440

Creating an RNAi feeding vector

AA

T T

Ligate PCR productinto T-tailed vector

T TA

A

Taq preferentially adds an extra “A” to the 3’ end of PCR products

L4440

L4440

This is a very efficient and useful process:

•Works for any PCR product

•The extra T on the vector inhibits vector self-ligation

How does the vector induce RNAi?

•Allows E. coli to produces double-stranded RNA

•Requires a specific strain of E. coli

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

T7 RNA polymerase

T7 RNA polymerase

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

Regulation of Lac Operon transcription

IPTG also induces

When IPTG is added T7 RNA polymerase is expressed

T7 RNA polymerase