Part:BBa_K3815011
Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
This is a section of V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in L4440 plasmid, and transformed into HT115(DE3).
purpose
We focused on feeding RNAi to kill these insects, based on previous studies showing that oral ingestion of dsRNA can induce RNAi-induced gene knockdown in Frankliniella occidentalis[1].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 452
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 320
cloning
This part was inserted in L4440. The L4440 has two t7 promoters, and this parts are transcribed from both sides.
production and purification methods
[[File:RNAgel.png|300px|thumb|right|Fig1.
(1 VATPase 2 VATPase 3 EPH1 4 L4440(vector) 5 VATPase1 6 VATPase2 7 EPH1 8 L4440(vector)
RNA was produced in 1L LB medium and eluted in 100ul of water.
1ug is based on the measurement results by nanodrop
We product ds RNA and purify this [2].
The 1L LB culture was started at 37C and IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours.
E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was collected by ethanol precipitation. After that, phenol-chloroform treatment was performed to purify RNA.
Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
We could confirm RNA production.
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