Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V004953 | pLP12 | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pLP12 is a novel suicide T-vector developed for rapid and efficient gene deletion in Vibrio species. To use pLP12, first, homologous arms (flanking sequences of the target gene) are amplified by PCR. These arms are directly ligated into AhdI-digested linearized pLP12. The recombinant plasmid is transformed into E. coli β2163 (a donor strain with efficient conjugation machinery, pir+) and transferred into Vibrio (pir-) via conjugation. Transconjugants with plasmid integration (first recombination) are selected on chloramphenicol-containing plates with D-glucose (to repress vmi480). For counterselection, these integrants are plated on medium with L-arabinose to induce vmi480, and surviving colonies (undergoing second recombination) are verified as target gene deletion mutants via PCR and sequencing.
- Vector Name:
- pLP12
- Antibiotic Resistance:
- Chloramphenicol
- Length:
- 3885 bp
- Type:
- Suicide vector
- Replication origin:
- R6K γ ori
- Source/Author:
- Luo P, He X, Liu Q, Hu C.
- Promoter:
- araBAD
- Growth Strain(s):
- DH5α-λpir
- Growth Temperature:
- 30℃
pLP12 vector Map
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
- Luo P, He X, Liu Q, Hu C. Developing Universal Genetic Tools for Rapid and Efficient Deletion Mutation in Vibrio Species Based on Suicide T-Vectors Carrying a Novel Counterselectable Marker, vmi480. PLoS One. 2015 Dec 7;10(12):e0144465. doi: 10.1371/journal.pone.0144465. PMID: 26641275; PMCID: PMC4671572.
pLP12 vector Sequence
LOCUS Exported 3885 bp DNA circular SYN 05-AUG-2025
DEFINITION synthetic circular DNA.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 3885)
TITLE Direct Submission
REFERENCE 2 (bases 1 to 3885)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..3885
/mol_type="other DNA"
/organism="synthetic DNA construct"
CDS 18..617
/codon_start=1
/label=vmi480
/translation="MMTKKPEFYAPDLTEERLHILSENLLDVLDEAHHYSESPNATAWF
KGTANYGLPQGMLIRMHSDSAYPWLTLANQTMDYTARVGNTLVQFVVDDPHSPRKQHRL
KRNAVEKHQISLELEEDYVDTPLIWRFYLNPISNGVDYSPSISLLGFNGNGNVICSWEY
DTVVTGPVVTDKPESVEIDEPLLVRKKKVQKKVSDE"
promoter complement(712..730)
/label=T3 promoter
/note="promoter for bacteriophage T3 RNA polymerase"
rep_origin 745..1133
/label=R6K gamma ori
/note="gamma replication origin from E. coli plasmid R6K;
requires the R6K initiator protein pi for replication"
CDS complement(1258..1917)
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
promoter complement(1918..2020)
/label=cat promoter
/note="promoter of the E. coli cat gene encoding
chloramphenicol acetyltransferase"
promoter 2147..2165
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
oriT 2319..2442
/label=incP origin of transfer
/note="incP origin of transfer"
CDS complement(2682..3560)
/codon_start=1
/gene="araC"
/product="L-arabinose regulatory protein"
/label=araC
/translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
EKVNDVAVKLS"
promoter 3587..3871
/gene="araBAD"
/label=araBAD promoter
/note="promoter of the L-arabinose operon of E. coli; the
araC regulatory gene is transcribed in the opposite
direction (Guzman et al., 1995)"