creating an rnai feeding vector how does ligation into l4440 work?
TRANSCRIPT
![Page 1: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/1.jpg)
Creating an RNAi feeding vector
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![Page 3: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/3.jpg)
How does ligation into L4440 work?
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Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
![Page 5: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/5.jpg)
Creating an RNAi feeding vector
AA
Taq preferentially adds an extra “A” to the 3’ end of PCR products
![Page 6: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/6.jpg)
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
![Page 7: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/7.jpg)
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
![Page 8: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/8.jpg)
Creating an RNAi feeding vector
AA
T T
Ligate PCR productinto T-tailed vector
T TA
A
L4440
L4440
![Page 9: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/9.jpg)
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
![Page 10: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/10.jpg)
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
![Page 11: Creating an RNAi feeding vector How does ligation into L4440 work?](https://reader035.vdocument.in/reader035/viewer/2022081515/56649e885503460f94b8be68/html5/thumbnails/11.jpg)
This is a very efficient and useful process:
•Works for any PCR product
•The extra T on the vector inhibits vector self-ligation
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How does the vector induce RNAi?
•Allows E. coli to produces double-stranded RNA
•Requires a specific strain of E. coli
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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
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T7 RNA polymerase
T7 RNA polymerase
E. coli strain HT115(DE3) has a modified lac promoter controlling the transcription of T7 RNA polymerase
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Regulation of Lac Operon transcription
IPTG also induces
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When IPTG is added T7 RNA polymerase is expressed
T7 RNA polymerase