pMTL-SC7315 vector (Cat. No.: V012834)

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Basic Information

Note: Plasmid vector pMTL-SC7315, used for allele exchange in C. difficile 630. Note that pMTL-SC7215 and pMTL-SC7315 are identical except that they have the pBP1 and pCB102 Gram-positive replicons, respectively (between AscI and FseI restriction sites).

Name:: pMTL-SC7315

Antibiotic Resistance:: Chloramphenicol

Length:: 6000 bp

Type:: C. difficile

Replication origin:: ori

Source/Author:: Cartman ST, Kelly ML, Heeg D, Heap JT , Minton NP

Copy Number:: High copy number

Growth Strain(s):: DH5alpha

Growth Temperature:: 37℃

$ 198.8

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

Cartman ST, Kelly ML, Heeg D, Heap JT, Minton NP. Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production. Appl Environ Microbiol. 2012 Jul;78(13):4683-90. doi: 10.1128/AEM.00249-12. Epub 2012 Apr 20. PMID: 22522680; PMCID: PMC3370502.