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Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids.
- 6 min
- Bacterial transformation steps. 1) Competent cell preparation, 2) transformation of cells, 3) cell recovery, and 4) cell plating. Competent cell preparation.
- Preparation of chemically competent and electrocompetent cells. Once prepared, competent cells should be evaluated for transformation efficiency.
- Bacterial transformation using (A) chemically competent cells and heat shock, and (B) electrocompetent cells and electroporation. Electroporation transformation.
- Bacterial transformation via electroporation. (A) Graph demonstrating exponential decay of electric pulse over time. (B) Workflow steps of the electroporation process.
This protocol explains how to transform a plasmid DNA template into competent bacterial cells which are then cultivated. The DNA is then puri ed from the cells using a miniprep kit. The competent cells used in this protocol come from the NEB®10-beta Competent E. coli kit.
Bacterial Transformation Protocols. Transformation is a key process in molecular cloning, by which multiple copies of recombinant DNA molecules are produced. The ability to take up free, extracellular genetic material is the prerequisite for bacterial competent cells to undergo transformation.
A comprehensive guide to bacterial transformation troubleshooting. Find solutions to obtain the ideal transformants.
May 21, 2012 · Overview. Quick Ligation products may be transformed by many different methods. The following protocol is recommended by New England Biolabs. Protocol. Thaw competent cells on ice. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube. Add 50 µl of competent cells to the DNA.
Using the transformation tube provided, 30 seconds at 42°C is optimal. Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes this step is shortened.
