Price Information
| Cat No. | Plasmid Name | Availability | Buy one, get one free! (?) |
|---|---|---|---|
| V008307 | pCold IV | In stock, instant shipping |
Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.
Basic Vector Information
pCold IV is a cold shock-induced expression vector that utilises the cspA promoter for efficient expression of recombinant proteins at 15°C, enhancing solubility and yield.
- Vector Name:
- pCold IV
- Antibiotic Resistance:
- Ampicillin
- Length:
- 4359 bp
- Type:
- Expression vector
- Replication origin:
- ori
- Source/Author:
- Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B, Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M.
- Promoter:
- cspA
- Growth Strain(s):
- DH10B
pCold IV 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
- Kondo S, Imura Y, Mizuno A, Homma M, Kojima S. Biochemical analysis of GTPase FlhF which controls the number and position of flagellar formation in marine Vibrio. Sci Rep. 2018 Aug 14;8(1):12115. doi: 10.1038/s41598-018-30531-5. PMID: 30108243; PMCID: PMC6092412.
pCold IV vector Sequence
LOCUS 40924_12695 4359 bp DNA circular SYN 17-DEC-2018
DEFINITION Expression vector pColdIV DNA, complete sequence.
ACCESSION .
VERSION .
KEYWORDS .
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4359)
AUTHORS Qing G, Ma LC, Khorchid A, Swapna GV, Mal TK, Takayama MM, Xia B,
Phadtare S, Ke H, Acton T, Montelione GT, Ikura M, Inouye M.
TITLE Cold-shock induced high-yield protein production in Escherichia coli
JOURNAL Nat. Biotechnol. 22 (7), 877-882 (2004)
PUBMED 15195104
REFERENCE 2 (bases 1 to 4359)
AUTHORS Takayama MM, Inouye M.
TITLE Direct Submission
JOURNAL Submitted (04-AUG-2004) Masanori Mitta Takayama, UMDNJ-Robert Wood
Johnson Medical School, Department of Biochemistry; 675 Hoes Lane,
Piscataway, NJ 08854-5635, USA (E-mail:mitsutam@takara-bio.co.jp,
Tel:81-77-543-7200, Fax:81-77-543-2494)
REFERENCE 3 (bases 1 to 4359)
TITLE Direct Submission
REFERENCE 4 (bases 1 to 4359)
AUTHORS .
TITLE Direct Submission
COMMENT SGRef: number: 1; type: "Journal Article"; journalName: "Nat.
Biotechnol."; date: "2004"; volume: "22"; issue: "7"; pages:
"877-882"
COMMENT SGRef: number: 2; type: "Journal Article"; journalName: "Submitted
(04-AUG-2004) Masanori Mitta Takayama, UMDNJ-Robert Wood Johnson
Medical School, Department of Biochemistry"; volume: " 675 Hoes
Lane, Piscataway, NJ 08854-5635, USA
(E-mail:mitsutam@takara-bio.co.jp, Tel:81-77-543-7200, Fax"; pages:
"81-77-543-2494"
COMMENT SGRef: number: 3; type: "Journal Article"
FEATURES Location/Qualifiers
source 1..4359
/mol_type="other DNA"
/organism="synthetic DNA construct"
promoter 15..81
/label=cspA promoter
/note="promoter of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
protein_bind 84..100
/label=lac operator
/note="The lac repressor binds to the lac operator to
inhibit transcription in E. coli. This inhibition can be
relieved by adding lactose or
isopropyl-beta-D-thiogalactopyranoside (IPTG)."
5'UTR 122..252
/label=cspA 5'UTR
/note="5'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
misc_feature 253..312
/gene="cspA"
/note="MCS = Multiple cloning sites contain the follow
restriction sites: NdeI, SacI, KpnI, XhoI, BamHI, EcoRI,
HindIII, SalI, PstI, XbaI"
3'UTR 320..464
/label=cspA 3'UTR
/note="3'UTR of the E. coli cold shock protein cspA gene
(Mitta et al., 1997)"
rep_origin complement(663..1118)
/direction=LEFT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
promoter 1218..1322
/label=AmpR promoter
CDS 1323..2180
/label=AmpR
/note="beta-lactamase"
rep_origin 2354..2942
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
protein_bind complement(3080..3101)
/label=CAP binding site
/note="CAP binding activates transcription in the presence
of cAMP."
CDS complement(3117..4196)
/label=lacI
/note="lac repressor"
promoter complement(4197..4274)
/label=lacI promoter