pNL1.2[NlucP] vector (Cat. No.: V011457)
Note: pNL1.2[NlucP] is a mammalian reporter vector encoding a secreted NanoLuc luciferase. It is designed for dual-luciferase assays, featuring a promoterless MCS for cloning regulatory elements. It is typically co-transfected with a control vector (e.g., firefly luciferase) for normalization.
- Name:
- pNL1.2[NlucP]
- Antibiotic Resistance:
- Ampicillin
- Length:
- 3233 bp
- Type:
- Luciferase Vectors
- Replication origin:
- ori
- Source/Author:
- Promega
- Copy Number:
- High copy number
Resources
Plasmid 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 ℃.
6. The concentration of plasmid re-measurement sometimes differs from the nominal value, which may be due to the position of the lyophilized plasmid in the tube, the efficiency of the re-dissolution, the measurement bias, and adsorption on the wall of the tube, therefore, it is recommended to transform and extract the plasmid before using it
General Plasmid Transform Protocol
1. Take one 100μl of the competent cells and thaw it on ice for 10min, add 2μl of plasmid, then ice bath for 30min, then heat-shock it at 42℃ for 60s, do not stir, and then ice bath for 2min.
2. Add 900μl of LB liquid medium without antibiotics, and incubate at 37℃ for 45min (30℃ for 1-1.5 hours) with 180rpm shaking.
3. Centrifuge at 6000rpm for 5min, leave only 100μl of supernatant to resuspend the bacterial precipitate and spread it onto the target plasmid-resistant LB plate.
4. Invert the plate and incubate at 37℃ for 14h, or at 30℃ for 20h.
5. Pick a single colony into LB liquid medium, add the corresponding antibiotics, incubate at 220rpm for 14h, and extract the plasmid according to the experimental needs and the instructions of the plasmid extraction kit.
References
- Ewunkem AJ, Deve M, Harrison SH, Muganda PM. Diepoxybutane induces the expression of a novel p53-target gene XCL1 that mediates apoptosis in exposed human lymphoblasts. J Biochem Mol Toxicol. 2020 Mar;34(3):e22446. doi: 10.1002/jbt.22446. Epub 2020 Jan 18. PMID: 31953984; PMCID: PMC7060116.
pNL1.2[NlucP] vector (Cat. No.: V011457) Sequence
LOCUS pNL1.2[NlucP]. 3233 bp DNA circular SYN 01-JAN-1980
DEFINITION Promoterless vector encoding destabilized NanoLuc(R) luciferase for
measuring the activity of promoter and enhancer sequences.
ACCESSION .
VERSION .
KEYWORDS pNL1.2[NlucP]
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3233)
AUTHORS Promega
TITLE Direct Submission
REFERENCE 2 (bases 1 to 3233)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3233
/mol_type="other DNA"
/organism="synthetic DNA construct"
misc_feature 1..70
/label=MCS
/note="MCS"
/note="multiple cloning site"
CDS 100..612
/label=Nluc
/note="NanoLuc(R) luciferase"
CDS 616..735
/label=hPEST
/note="PEST degradation sequence from mouse ornithine
decarboxylase"
polyA_signal complement(787..908)
/label=SV40 poly(A) signal
/note="SV40 polyadenylation signal"
rep_origin complement(1327..1915)
/direction=LEFT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
CDS complement(2118..2975)
/label=AmpR
/note="beta-lactamase"
polyA_signal 3080..3128
/label=poly(A) signal
/note="synthetic polyadenylation signal"
misc_feature 3142..3233
/label=pause site
/note="RNA polymerase II transcriptional pause signal from
the human alpha-2 globin gene"