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

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

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

Creating an RNAi feeding vector
A
A
Ligate PCR product into T-tailed vector
L4440
L4440

Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
A
A
Ligate PCR product into T-tailed vector
L4440
L4440

Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
A
A
Ligate PCR product into T-tailed vector
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

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

IPTG also induces

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