pBluescript II KS(-) vector (Cat. No.: V012545)

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Basic Information

Note: pBluescript II KS(-) is a high-copy-number phagemid cloning vector (2961 bp) with ampicillin resistance. It features T3 and T7 promoters flanking a multiple cloning site (MCS) for gene manipulation and an f1 origin for producing single-stranded DNA, commonly used for sequencing and in vitro transcription.

Name:: pBluescript II KS(-)

Antibiotic Resistance:: Ampicillin

Length:: 2961 bp

Type:: Cloning Vectors

Replication origin:: ori

Source/Author:: Alting-Mees MA, Short JM.

Copy Number:: High copy number

5' Primer:: M13 fwd

3' Primer:: M13 rev

Growth Temperature:: 37℃

$ 138.5

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Two tubes of lyophilized plasmid will be delivered, each tube is about 5µg.

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

Somyoonsap P, Kitpreechavanich V, Pornbanlualap S. A sequence-specific nicking endonuclease from streptomyces: purification, physical and catalytic properties. ISRN Biochem. 2013 Aug 21;2013:287158. doi: 10.1155/2013/287158. PMID: 25937959; PMCID: PMC4392989.

pBluescript II KS(-) vector (Cat. No.: V012545) Sequence

LOCUS       40924_6776        2961 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Standard cloning vector (phagemid excised from lambda ZAPII). The f1
            (–) orientation allows rescue of antisense strand ssDNA. pBluescript
            II SK(–) has a reversed MCS.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2961)
  AUTHORS   Alting-Mees MA, Short JM.
  TITLE     pBluescript II: gene mapping vectors.
  JOURNAL   Nucleic Acids Res. 1989;17:9494.
  PUBMED    2555794
REFERENCE   2  (bases 1 to 2961)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 2961)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "1989"; volume: "17"; pages: "9494"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2961
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    653..760
                     /label=MCS
                     /note="pBluescript multiple cloning site"
     promoter        complement(773..791)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(812..828)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(836..852)
                     /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)."
     promoter        complement(860..890)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(905..926)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(1214..1802)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(1976..2833)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(2834..2938)
                     /label=AmpR promoter