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Transformation. Add between 1-5 SL of ligation mixture to competent cells for transformation. Extended ligation with PEG causes a drop-off in transformation efficiency (Quick Ligation Kit). Electroporating is recommended for large constructs (>10,000 BP).
The optimal amount of DNA is lower than commonly recognized. Using clean, super coiled pUC19, the efficiency of transformation is highest in the 100 pg-1 NG range. However, the total colonies which can be obtained from a single transformation reaction increase up to about 100 NG.
For successful chemical transformation, 50100 SL of competent cells and 110 NG of DNA are recommended. When a ligation mixture is used as the transforming DNA (often 15 All is sufficient), purification prior to chemical transformation is generally not required.
Once DNA is added to the cells, electroporating 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 SL.
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transform ants by the amount of DNA used during a transformation procedure.
A good rule to follow is this: if your efficiency is equal to or less than 5 × 107 CFU/kg DNA, use these cells for plasmid transformations. If your efficiency is greater than 5 × 107 (ideally 1 × 108 or higher), use these cells for ligation and other assembly reaction transformations.
In a typical cloning experiment, researchers first insert a piece of DNA, such as a gene, into a circular piece of DNA called a plasmid. This step uses restriction enzymes and DNA ligase and is called a ligation. After a ligation, the next step is to transfer the DNA into bacteria in a process called transformation.
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiesterase bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication.
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