pFA6a-kanMX6 vector (Cat. No.: V010292)

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Basic Information

Note: pFA6a-kanMX6 is a yeast shuttle vector designed for PCR-based gene targeting. It contains a kanMX6 module (conferring G418 resistance in yeast) driven by the A. gossypii TEF promoter/terminator, an ampicillin resistance gene for bacterial selection, and is used for gene deletion or modification in yeast via homologous recombination.

Name:: pFA6a-kanMX6

Antibiotic Resistance:: Ampicillin

Length:: 3938 bp

Type:: Yeast Plasmids

Replication origin:: ori

Host:: Yeast

Source/Author:: Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, Brachat A,

Copy Number:: High copy number

Promoter:: TEF

Growth Temperature:: 37℃

$ 99.4

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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 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

Bähler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 1998 Jul;14(10):943-51. doi: 10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y. PMID: 9717240.