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pRSET B vector (V012417)

Price Information

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V012417 pRSET B In stock, instant shipping

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

Basic Vector Information

pRSET B is a high-level prokaryotic expression controlled by the strong bacteriophage T7 promoter.

Vector Name:
pRSET B
Antibiotic Resistance:
Ampicillin
Length:
2887 bp
Type:
Prokaryotic expression vector
Replication origin:
ori
Source/Author:
Invitrogen (Life Technologies)
Copy Number:
High copy number
Growth Strain(s):
Stbl3
Growth Temperature:
37℃

pRSET B vector Map

Copy Sequence View Map
pRSET B2887 bp600120018002400T7 promoterRBSATG6xHisT7 tag (gene 10 leader)MCST7 terminatorf1 oriAmpR promoterAmpRori

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

  • Tan MP, Mohamed Alitheen NB, Tan WS, Yap WB. Expression of Influenza M2e-NP Recombinant Fusion Protein in Escherichia coli BL21 (DE3) and Its Binding to Antibodies. Vaccines (Basel). 2022 Dec 1;10(12):2066.

pRSET B vector Sequence

LOCUS       pRSET_B.        2887 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Bacterial vector for high-level expression of proteins with a 
            cleavable 6xHis tag. For other reading frames, use pRSET A or pRSET 
            C.
ACCESSION   .
VERSION     .
KEYWORDS    pRSET B
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2887)
  AUTHORS   Invitrogen (Life Technologies)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 2887)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..2887
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     RBS             70..92
                     /label=RBS
                     /note="efficient ribosome binding site from bacteriophage
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..102
                     /codon_start=1
                     /product="start codon"
                     /label=start codon
                     /note="ATG"
                     /translation="M"
     CDS             112..129
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             133..165
                     /label=T7 tag (gene 10 leader)
                     /note="leader peptide from bacteriophage T7 gene 10"
     CDS             169..192
                     /label=Xpress(TM) tag
                     /note="Xpress(TM) epitope tag, including an enterokinase 
                     recognition and cleavage site"
     misc_feature    192..238
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     terminator      302..349
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      446..901
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        927..1031
                     /label=AmpR promoter
     CDS             1032..1889
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      2063..2651
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"