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XL10 competent cell Catalog # C503 Introduction This product is super competent E.coli XL10 cell for chemical transformation with transformation efficiency greater than 108 cfu/ μg. It can be widely used in plasmid transformation, gene cloning and blue-white screening. Package information Component C503-01 C503-02 C503-03 XL10 competent cell* 5× 100 μl 10× 100 μl 20× 100 μl Genetype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lacTet r Hte [F′proAB lacIqZΔM15 Tn10(Tet r) Amy Cam r] Storage Stored at -80℃. Do not store the product at -20℃ or in liquid nitrogen. Advantages - XL10 strain of E.coli is a kind of new strain for gene cloning with tetracycline (Tet r) and chloramphenicol (Cam r) resistance. - Applicable to high efficient transformation of PCR product, cDNA and other unmethylated DNA. - EndA1 and RecA1 mutation guarantee the stability of exogenous DNA in the host bacteria, which is conductive to DNA extraction to get high-quality plasmid. - Due to the presence of the Hte genotype, the transformation efficiency of large fragments of DNA is greater, and the growth of bacteria is greatly improved. - Applicable to blue-white screening of recombinant plasmids. - Transformation efficiency ≥ 108 cfu/ μg Puc19 DNA. Quality Control No exogenous residual plasmid DNA Transformation efficiency ≥ 108 cfu/ μg Puc19 DNA Protocols 1. Remove the competent cells from refrigerator of -80℃, and place the competent cells in an ice water bath to melt. 2. The DNA is added into 100μl competent cells, mix gently. Keep in the ice bath for 30 min. 3. Incubate cells for 90 sec. at 42℃. And return to the ice bath for 2~3 min. 4. Add SOC or LB medium up to a final volume of 1 ml. keep in water bath for 10 min at 37℃. 5. Incubate by shaking (150 rpm) for 45 min at 37℃ to recovery and gain resistance. 6. Centrifuged at 2500g for 5 min, and remove 900 μl supernatant, mix gently. And plate on selective media. Incubate overnight at 37℃. Notes 1. 2. 3. 4. The competent cells should be used immediately after thawed in an ice water bath. The volume of DNA sample should not exceed 1/10 of the volume of competent cells. After added the DNA sample, the competent cell should not be pipetting, and just mix gently. To ensure the high transformation efficiency, the whole operation should be gentle in cool environment. Appendix I: the working concentration of common antibiotic. Antibiotic Working concentration Ampicillin Carbenicillin Chloramphenicol Kanamycin Streptomycin Tetracycline 100 μg/ml 100 μg/ml 33 μg/ml 30 μg/ml 25 μg/ml 15 μg/ml Appendix II: the transformation inhibitors in DNA sample and removal methods Inhibitor of transformation Removal methods Detergents Phenol Ethanol or isopropanol PEG Ethanol precipitation Chloroform extraction and ethanol precipitation Dry thoroughly before DNA is dissolved Column purification or Chloroform extraction and ethanol precipitation Column purification or Chloroform extraction and ethanol precipitation DNA binding proteins