Difference between revisions of "Part:BBa K3815011"
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
−
<partinfo>BBa_K3815011 short</partinfo>
+
<partinfo>BBa_K3815011 short</partinfo><br>
−
ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in L4440 plasmid, and transformed into HT115(DE3).
+
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).
 
<h3><font size="3"></font> purpose </h3>
 
<h3><font size="3"></font> purpose </h3>
 
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].  
 
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].  
Line 18: Line 18:
  
  
−
RNA purification was followed this protocol[2].
 
 
[[File:RNAgel.png|300px|thumb|right|Fig1. RNA]]
 
[[File:RNAgel.png|300px|thumb|right|Fig1. RNA]]
−
[[File:RNAgel2.png|300px|thumb|right|Fig1. RNA2]]
+
 
−
1. Add 1 ml of ampicillin per 1 L of autoclaved LB. <br>
+
I 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. <br>
−
2.Spread 3ml of LB on a plate, suspend the colonies, and return to LB. <br>
+
 
−
3.Start incubation at 37℃.(180rpm)<br>
+
Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
−
4.After incubation, when the OD reaches 0.4, add 200ul of 1M IPTG and induce RNA production with 0.2mM IPTG.<br>
+
 
−
5.After the addition of IPTG, the culture was continued at 37c for 4h, and then the precipitates were collected.<br>
+
−
6.The collected precipitates were suspended in 5ml 70%Etoh in PBS.<br>
+
−
7.4c 5 min incubate<br>
+
−
8.Centrifuge at 10000 rpm for 10 min <br>
+
−
9.Drain off supernatant<br>
+
−
10.Suspend the precipitates in 3ml 150mM NaCl<br>
+
−
11.4c 1h incubate<br>
+
−
12.10min 10000 rpm centrifuge<br>
+
−
13.Collect supernatant (also freeze precipitate at -20c)<br>
+
−
14.Add 8ml of 100% EtOH to supernatant<br>
+
−
15.-20c overnight<br>
+
−
16.centrifuge at 4C 10000rpm for 30min.<br>
+
−
17.Discard the supernatant.<br>
+
−
18.Wash the precipitates with 1ml of 85% EtOh.<br>
+
−
19.Centrifuge at 4c 10000rpm for 3min.<br>
+
−
20.Discard the supernatant<br>
+
−
21.4c 10000rpm 1min centrifugation<br>
+
−
22.Discard the supernatant.<br>
+
−
23.Elute the precipitates into 100ul of DW.<br>
+
−
24.The amount of RNA estimated by PAGE electropharased .<br>
+
  
 
<br>
 
<br>

Revision as of 06:09, 21 October 2021


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 452
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 320


production and purification methods


Fig1. RNA
I 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.