Creating an RNAi feeding vector

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Creating an RNAi feeding vector
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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
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
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
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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
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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
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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
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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
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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
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• 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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E. coli strain HT115(DE3) has a modified lac
promoter controlling the transcription of T7 RNA
polymerase
T7 RNA polymerase
T7 RNA polymerase
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Regulation of Lac Operon transcription
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When IPTG is added T7 RNA polymerase is expressed
T7 RNA polymerase