[Direkt zum Inhalt](https://www.sigmaaldrich.com#main-content) [![Merck](https://www.sigmaaldrich.com/static/logos/purple/merck.svg)](https://www.sigmaaldrich.com/DE/de) Produkte Warenkorb0 DEDE Produkte ProdukteAnwendungenDienstleistungenRessourcenUnterstützung [Anmelden / Registrieren](https://www.sigmaaldrich.com/oidc-sign-in) [Bestellungssuche](https://www.sigmaaldrich.com/DE/de/order-lookup) [Schnelleinkauf](https://www.sigmaaldrich.com/DE/de/quick-order) Warenkorb0 [Home](https://www.sigmaaldrich.com/DE/en)[Cloning & Expression](https://www.sigmaaldrich.com/DE/en/applications/genomics/cloning-and-expression)Preparing Cells for Electroporation # Preparing Cells for Electroporation Cell electroporation is a method that uses an electrical field to increase cell membrane permeability so DNA or other substance can be introduced into the cell. ## Reagent Preparation for Cell Electroporation 2× YT medium:Dissolve 16 g of tryptone, 10 g of yeast extract, and 5 g of NaCl in 900 mL of distilled water. Adjust the pH to 7.0 with NaOH. Adjust the volume to 1 L with distilled water. Sterilize by autoclaving for 20 min. To prepare as a solid medium, add 1.2% to 1.5% agar. 1 mM HEPES:0.26 g of HEPES, sodium salt. Dissolve in 900 mL of distilled water. Adjust the pH to 7.0. Adjust the volume to 1 L with distilled water. Sterilize by autoclaving. 10% glycerol in 1 mM HEPES, pH 7.0:Aseptically add 10 mL of sterile 100% glycerol to 90 mL of sterile 1 mM HEPES, pH 7.0. 10% glycerol in distilled water:Add 10 mL of 100% glycerol to 90 mL of distilled water. Sterilize by autoclaving. Isopropanol TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA Phenol:Redistilled phenol saturated with TE buffer containing 8-hydroxy quinoline Chloroform/isoamyl alcohol:Reagent-grade chloroform and isoamyl alcohol, mixed 24:1 Phenol/chloroform:Equal parts of redistilled phenol and chloroform/isoamyl alcohol (24:1), each prepared as described above 3 M sodium acetate, pH 5.4, aqueous solution Ethanol, 70%, 95% Table 1.Reagents Required ## Procedure 01. Inoculate 10 mL of 2× YT medium with an *E. coli* host strain from an LB or 2× YT medium plate. Incubate at 37 °C overnight with shaking. 02. Inoculate 1 L of 2× YT medium with the 10 mL of an overnight culture of host cells. Incubate for 2 to 2.5 h at 37 °C with shaking at 250 rpm until an A600 of 0.5 to 0.7 is achieved. 03. Place the flask on ice for 15 to 30 min. 04. Spin at 4,000 × g for 20 min at 4 °C. 05. Decant the supernatant and resuspend the cells in 1 L of ice-cold sterile 1 mM HEPES, pH 7.0. 06. Spin as described above. Decant the supernatant and resuspend the cells in 500 mL of ice-cold sterile 1 mM HEPES, pH 7.0. 07. Spin as described above. Decant the supernatant. Wash the cells in 20 mL of sterile 1 mM HEPES, pH 7.0, containing 10% glycerol. 08. Spin as described above. Decant the supernatant. Resuspend the cells in a total volume of 2 to 3 mL of sterile 10% glycerol in distilled water. 09. Dispense in 50 to 100 µL aliquots and proceed to the electroporation protocol or freeze on dry ice and store at -70 °C. 10. Extract the ligated pGEX vector (as well as the uncut vector) once with an equal volume of phenol/chloroform and once with an equal volume of chloroform/isoamyl alcohol. 11. Remove the aqueous phase and add 1/10 volume of 3 M sodium acetate, pH 5.4 and 2.5 volumes of 95% ethanol. 12. Place on dry ice for 15 min and then spin in a microcentrifuge for 5 min to pellet the DNA. 13. Remove the supernatant and wash the pellet with 1 mL of 70% ethanol. Centrifuge for 5 min, discard the supernatant, and dry the pellet. 14. Resuspend each DNA pellet in 20 µL of sterile distilled water. Alternatively, the DNA can be gel band-purified. #### Note: The DNA must be completely free of salt before electroporation. ## Electroporation Efficiency One nanogram of uncut (supercoiled) vector DNA is recommended to be transformed in parallel with insert/pGEX ligations to determine the efficiency of each competent cell preparation. For more information about electroporation protocols, see the instructions for the selected electroporation system. ## Materials Bitte entschuldigen Sie, es ist ein unerwarteter Fehler aufgetreten Response not successful: Received status code 500 __Related Articles__ - [Introduction to Cell Transfection](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/transfection-reagents) - [Reverse Transfection of Plasmid DNA](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/cell-culture-and-cell-culture-analysis/transfection-and-gene-editing/xtg-reverse-transfect-pdna) - [3xFLAG® System Expression Vectors for Ultra-Sens](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/3x-flag) - [Blue-White Screening & Protocols for Colony Selection](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/blue-white-screening) - [Bacterial Transformation Protocols](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/competent-cells) - [Genotypes, Phenotypes and Markers](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/definitions-and-genotypes-of-e-coli) - [Restriction Enzyme Cloning Glossary](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/estriction-enzyme-cloning-manual-glossary) - [How Transfection Works](https://www.sigmaaldrich.com/DE/en/technical-documents/technical-article/genomics/cloning-and-expression/how-transfection-works) Oben __Melden Sie sich an, um fortzufahren.__ Um weiterzulesen, melden Sie sich bitte an oder erstellen ein Konto. 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