Electroporation cuvettes and microcentrifuge tubes should be pre-chilled on ice.
Electrocompetent cells should be thawed on ice and suspended well by carefully flicking the tubes.
Once DNA is added to the cells, electroporation can be carried out immediately. It is not necessary to incubate DNA with cells. The maximum recommended volume of a DNA solution to be added is 2.5 µl. Addition of a large volume of DNA decreases transformation efficiency.
Contaminants such as salts and proteins can lower electroporation efficiency. Ideally, DNA for transformation should be purified and suspended in water or TE. Transformation efficiency is more than10-fold lower for ligation mixtures than the control pUC19 plasmid due to the presence of ligase and salts. If used directly, ligation reactions should be heat-inactivated at 65°C for 20 min and then diluted 10-fold. For optimal results, spin columns (NEB #T1030) are recommended for cleanup of ligation reactions.
Electroporation conditions vary with different cuvettes and electroporators. If you are using electroporators or cuvettes not specified in the protocol, you may need to optimize the electroporation conditions. Cuvettes with 1mm gap are recommended (e.g. BTX Model 610/613 and Bio-Rad #165-2089). Higher voltage is required for cuvettes with 2 mm gap.
Arcing may occur due to high concentration of salts or air bubbles.
It is essential to add recovery medium to the cells immediately after electroporation. One minute delay can cause a 3-fold reduction in efficiency.
Cold and dry selection plates lead to lower transformation efficiency. Pre-warm plates at 37°C for 1 hour. Using 37°C pre-warmed recovery medium increases the efficiency by about 20%.
Refreeze unused cells in a dry ice/ethanol bath for 5 min and then store at -80°C. Do not use liquid nitrogen. Additional freeze-thaw cycles result in lower transformation efficiency.
NEB® 10-beta Electrocompetent E. coli
NEB® Turbo Competent E. coli (High Efficiency)
SOC Outgrowth Medium
phi29 DNA Polymerase
Monarch® Plasmid Miniprep Kit
Monarch® DNA Gel Extraction Kit
Monarch® PCR & DNA Cleanup Kit (5 μg)
STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
操作说明、说明书 & 用法
Electroporation Protocol (C2986)
Additional E. coli Strain Genotypes
McrA, McrBC and EcoKI Strain Phenotypes
Traditional Cloning Quick Guide
工具 & 资源
Characteristics of Select E.coli Strains
Competitor Cross-Reference Tool
FAQs & 问题解决指南
How should I calculate the Electrotransformation efficiency (C2986)?
What are the solutions/recipes (C2986)?
What are the strain properties (C2986)?
Can any NEB competent cells help with the generation of custom phage libraries? An F factor is required for such libraries.
How should I store SOC Outgrowth Medium? The SOC I received with my competent cells recommends storage at either room temperature or 4°C, however, when I purchase it as a stand alone product, it recommends storing it at 4°C. Which is better?
How should fragments be prepared for assembly using NEBuilder HiFi?
Are NEB Turbo Competent E. coli (High Efficiency) or NEB Turbo Electrocompetent E. coli recommended for cloning repetitive or unstable DNA?