pMAD vector (V007385)

Price Information

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

Basic Vector Information

A shuttle vector named pMAD was developed to rapidly generate gene inactivation mutants in naturally nontransformable Gram-positive bacteria. This vector enables swift colorimetric blue-white screening on X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) plates, which simplifies the identification of bacteria that have lost the plasmid, thus streamlining the cloning process during mutagenesis. The plasmid was effectively employed in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to create mutants, with or without an accompanying antibiotic resistance gene.

pMAD 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

• Arnaud M, Chastanet A, Débarbouillé M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl Environ Microbiol. 2004;70(11):6887-6891. doi:10.1128/AEM.70.11.6887-6891.2004

pMAD vector Sequence

LOCUS       Exported                9664 bp DNA     circular SYN 05-OCT-2024
DEFINITION  Exported.
ACCESSION   V007385
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9664)
  TITLE     Direct Submission
REFERENCE   2  (bases 1 to 9664)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9664)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9664
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(98..829)
                     /gene="ermC"
                     /label=rRNA adenine N-6-methyltransferase
                     /note="rRNA adenine N-6-methyltransferase from
                     Staphylococcus aureus. Accession#: P02979"
     CDS             1241..2449
                     /gene="pre"
                     /label=Plasmid recombination enzyme type 1
                     /note="Plasmid recombination enzyme type 1 from
                     Staphylococcus aureus. Accession#: P03857"
     CDS             complement(3973..5988)
                     /gene="bgaB"
                     /label=Beta-galactosidase bgaB
                     /note="Beta-galactosidase bgaB from Geobacillus
                     kaustophilus. Accession#: P19668"
     CDS             7221..7409
                     /label=rop
                     /note="Rop protein, which maintains plasmids at low copy
                     number"
     misc_feature    7514..7654
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(7840..8428)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8602..9459)
                     /label=AmpR
                     /note="beta-lactamase"
     promoter        complement(9460..9564)
                     /label=AmpR promoter