pLac-maoA(opt-ecoli1917)-HA vector (V014825)

Price Information

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

Basic Vector Information

pLac-maoA(opt-ecoli1917)-HA 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.

pLac-maoA(opt-ecoli1917)-HA vector Sequence

LOCUS       V014825                 4656 bp    DNA     circular SYN 01-JAN-1980
DEFINITION  Exported.
ACCESSION   V014825
VERSION     V014825
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
            .
REFERENCE   1  (bases 1 to 4656)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..4656
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(260..805)
                     /direction=LEFT
                     /label="p15A ori"
                     /note="Plasmids containing the medium-copy-number p15A
                     origin of replication can be propagated in E. coli cells
                     that contain a second plasmid with the ColE1 origin."
     protein_bind    935..956
                     /label="CAP binding site"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        971..1001
                     /label="lac promoter"
                     /note="promoter for the E. coli lac operon"
     CDS             1046..3316
                     /gene="tynA"
                     /label="Primary amine oxidase"
                     /note="Primary amine oxidase from Escherichia coli (strain
                     K12). Accession#: P46883"
     CDS             3317..3343
                     /label="HA"
                     /note="HA (human influenza hemagglutinin) epitope tag"
     CDS             complement(3527..4333)
                     /label="KanR"
                     /note="aminoglycoside phosphotransferase"