Difference between revisions of "Part:BBa K3815011"
| Line 20: | Line 20: | ||
4.After incubation, when the OD reaches 0.4, add 200ul of 1M IPTG and induce RNA production with 0.2mM IPTG.<br> | 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> | 5.After the addition of IPTG, the culture was continued at 37c for 4h, and then the precipitates were collected.<br> | ||
| − | The collected precipitates were suspended in 5ml 70%Etoh in PBS.<br> | + | 6.The collected precipitates were suspended in 5ml 70%Etoh in PBS.<br> |
| − | 4c 5 min incubate<br> | + | 7.4c 5 min incubate<br> |
| − | Centrifuge at 10000 rpm for 10 min <br> | + | 8.Centrifuge at 10000 rpm for 10 min <br> |
| − | Drain off supernatant<br> | + | 9.Drain off supernatant<br> |
| − | Suspend the precipitates in 3ml 150mM NaCl<br> | + | 10.Suspend the precipitates in 3ml 150mM NaCl<br> |
| − | 4c 1h incubate<br> | + | 11.4c 1h incubate<br> |
| − | 10min 10000 rpm centrifuge<br> | + | 12.10min 10000 rpm centrifuge<br> |
| − | Collect supernatant (also freeze precipitate at -20c)<br> | + | 13.Collect supernatant (also freeze precipitate at -20c)<br> |
| − | Add 8ml of 100% EtOH to supernatant<br> | + | 14.Add 8ml of 100% EtOH to supernatant<br> |
| − | -20c overnight<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> | |
| − | The amount of RNA | + | |
<br> | <br> | ||
Revision as of 16:18, 20 October 2021
Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
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
production and purification methods
1. Add 1 ml of ampicillin per 1 L of autoclaved LB.
2.Spread 3ml of LB on a plate, suspend the colonies, and return to LB.
3.Start incubation at 37℃.(180rpm)
4.After incubation, when the OD reaches 0.4, add 200ul of 1M IPTG and induce RNA production with 0.2mM IPTG.
5.After the addition of IPTG, the culture was continued at 37c for 4h, and then the precipitates were collected.
6.The collected precipitates were suspended in 5ml 70%Etoh in PBS.
7.4c 5 min incubate
8.Centrifuge at 10000 rpm for 10 min
9.Drain off supernatant
10.Suspend the precipitates in 3ml 150mM NaCl
11.4c 1h incubate
12.10min 10000 rpm centrifuge
13.Collect supernatant (also freeze precipitate at -20c)
14.Add 8ml of 100% EtOH to supernatant
15.-20c overnight
16.centrifuge at 4C 10000rpm for 30min.
17.Discard the supernatant.
18.Wash the precipitates with 1ml of 85% EtOh.
19.Centrifuge at 4c 10000rpm for 3min.
20.Discard the supernatant
21.4c 10000rpm 1min centrifugation
22.Discard the supernatant.
23.Elute the precipitates into 100ul of DW.
24.The amount of RNA estimated by PAGE electropharased .