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DH5α-i strain is the most commonly used competent cell in the laboratory.
DH5α-i is a new type of competent cell optimized for pD2P plasmid vector, which can further improve the transformation efficiency and extraction yield of pD2P plasmid DNA.
It is better to use it in combination with ProteinFactory cell-free protein synthesis products. Deletion of endonuclease (endA) and recombinase deficient (recA) reduce the homologous recombination probability of inserted fragments and ensure the stability of inserted DNA. The existence of lacZΔM15 makes DH5α-i can be used for blue-white screening. DH5α-i competent cells were prepared by special process, and the pUC19 plasmid detection transformation efficiency was > 108 cfu/μg DNA.
1. Take out the competent cells from the -80 ℃ refrigerator, and melt them in an ice bath. Absorb 100 μL competent cells and transfer to a shake tube pre-cooled overnight at -20 ℃, add the pD2P plasmid or ligation products and mix gently, then ice bath for 30 min.
2. Heat shock in -42 ℃ ice bath for 60 s, quickly put it back into the ice and stand still for 2 min. Do not shake the shake tube in this process.
3. Add 900 μL SOC or LB medium warm-bathed in 37 ℃ to the shake tube (SOC medium can improve the transformation efficiency by 2-3 times). Put into 37 ℃, 180 rpm, 45 min to revive the strain.
4. According to the experimental requirements (transformation of pD2P plasmid and recombinant ligation products), absorb different volumes of transformed competent cells and add them to LB agar medium containing corresponding antibiotics. Evenly spread the cells, and place the plate at 37 ℃ until the liquid is absorbed, then invert the plate and culture at 37 ℃ overnight. If the blue-white screening operation is required, place the plate at 37 ℃ for at least 15 h to culture.
Note: When kanamycin and tetracycline are used as screening resistance, they need to be resuscitated in SOC medium to improve the transformation efficiency. When ampicillin is used as screening resistance, this step can be omitted. After the competent cell DNA mixture is incubated on ice for 5-10 min, add 4 times volume of SOC medium warm-bathed in 37 ℃, then leave in 37 ℃, 180 rpm for 1 h incubation, and then transfer to 37 ℃ preheated LB medium.
1. Pre-heat the screening medium plate at 37 ℃, 15 min in advance.
2. Take out the DH5α-i competent cells from -80 ℃ refrigerator, quickly insert them into the ice, wait 5 min for the bacteria to melt, add the target DNA (plasmid or ligation products) and wobble the EP tube bottom by hand to mix them gently, and stand still in the ice for 5 min.
3. Use 200 μL pipette to transfer the competent cell-DNA mixture to LB medium preheated at 37 ℃, then apply it evenly and make sure no water stain left on the surface.
4. Place the plate upside down in a 37 ℃ incubator overnight for culture. If the blue-white screening is required, place the plate at 37 ℃ for at least 15 h to culture.
1. The freshly thawed cells produce the highest conversion efficiency.
2. Avoid repeated thawing.
3. Avoid pipette blowing.
4. The whole operation should be gentle.
5. The 10-minute rapid transformation method is not recommended for the recombinant product.
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