Price Information
Cat No. | Plasmid Name | Availability | Add to cart |
---|---|---|---|
V012784 | L4440 | In stock (lyophilized plasmid) |
Buy one, get one free! |
Two vials of lyophilized plasmid will be delivered, each vial is about 5µg.
Basic Vector Information
E. coli bacteria carrying RNAi plasmids that produce the desired dsRNA can be fed to worms, and the dsRNA is transferred to the worm via the intestinal tract. RNAi plasmids typically consist of DNA coding sequence from the intended target gene cloned between two T7lac promoters. The plasmid also has a selectable marker that confers resistance to an antibiotic, in this case ampicillin. In 7.15, you will use the E. coli strain HT115 carrying various L4440 plasmids, each containing a different cloned gene sequence. HT115 is an RNase III-deficient E. coli strain with IPTG-inducible T7 polymerase activity. To induce dsRNA production from these plasmids, the HT115 bacteria is grown on special RNAi NGM feeding plates that contain IPTG and the ampicillin analog carbenicillin. Carbenicillin is preferred over ampicillin because it tends to be more stable.
- Vector Name:
- L4440
- Antibiotic Resistance:
- Ampicillin
- Length:
- 2790 bp
- Type:
- RNAi
- Replication origin:
- ori
- Source/Author:
- Andrew Fire
- Copy Number:
- High copy number
- Promoter:
- T7
- Cloning Method:
- Enzyme Cut
- Growth Strain(s):
- stbl3
- Growth Temperature:
- 37℃
- Expression Method:
- IPTG induced
L4440 vector Vector Map
Plasmid Resuspension Protocol:
1. Centrifuge at 5,000×g for 5 min.
2. Carefully open the tube and add 20 μl of sterile water to dissolve the DNA.
3. Close the tube and incubate for 10 minutes at room temperature.
4. Briefly vortex the tube and then do a quick spin to concentrate the liquid at the bottom. Speed is less than 5000×g.
5.Store the plasmid at -20 ℃.
References
- Chen HH, Dewer Y, Wang Y, Tan SQ, Liu XL, Shi WP. Interference with orco gene expression affects host recognition in Diorhabda tarsalis. Front Physiol. 2022 Dec 20;13:1069391. doi: 10.3389/fphys.2022.1069391.
L4440 vector Sequence
LOCUS 40924_1584 2790 bp DNA circular SYN 23-SEP-2021 DEFINITION synthetic circular DNA. ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 2790) TITLE Direct Submission REFERENCE 2 (bases 1 to 2790) AUTHORS . TITLE Direct Submission COMMENT SGRef: number: 1; type: "Journal Article" FEATURES Location/Qualifiers source 1..2790 /mol_type="other DNA" /organism="synthetic DNA construct" promoter 19..37 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind 95..111 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind complement(181..197) /label=KS primer /note="common sequencing primer, one of multiple similar variants" promoter complement(223..241) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(251..267) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" rep_origin 409..864 /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" promoter 890..994 /label=AmpR promoter CDS 995..1852 /codon_start=1 /label=AmpR /note="beta-lactamase" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" rep_origin 2026..2614 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication"