pT7Blue vector (Cat. No.: V012384)

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Basic Information

Note: pT7Blue serves as the backbone plasmid for detecting the restriction endonuclease activity of UpaP162 and the protective effect of the methyltransferase UPV229 in Ureaplasma parvum.

Name:: pT7Blue

Antibiotic Resistance:: Ampicillin

Length:: 2887 bp

Type:: Cloning Vectors

Replication origin:: ori

Source/Author:: Novagen (EMD Millipore)

Copy Number:: High copy number

5' Primer:: M13 fwd

3' Primer:: M13 rev

Growth Strain(s):: DH10B

$ 199.1

In stock, instant shipping

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 sterile water to dissolve the DNA: add 20 μl for 5 μg plasmid, and 100 μl for 100 μg plasmid.

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

Wu HN, Nakura Y, Yoshimura M, Gaddi Tantengco OA, Nomiyama M, Takayanagi T, Fujita T, Yasukawa K, Yanagihara I. Type II restriction modification system in Ureaplasma parvum OMC-P162 strain. PLoS One. 2018 Oct 16;13(10):e0205328. doi: 10.1371/journal.pone.0205328. PMID: 30325937; PMCID: PMC6191088.

pT7Blue vector (Cat. No.: V012384) Sequence

LOCUS       pT7Blue.        2887 bp DNA     circular SYN 01-JAN-1980
DEFINITION  Cloning vector with a T7 promoter, and with a modified multiple 
            cloning site that includes an EcoRV site for blunt cloning or TA 
            cloning.
ACCESSION   .
VERSION     .
KEYWORDS    pT7Blue
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2887)
  AUTHORS   Novagen (EMD Millipore)
  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        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    45..125
                     /label=MCS
                     /note="MCS"
                     /note="multiple cloning site"
     primer_bind     complement(126..142)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      complement(284..739)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        766..870
                     /label=AmpR promoter
     CDS             871..1728
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      1902..2490
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     protein_bind    2778..2799
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        2814..2844
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2852..2868
                     /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)."