How to Find Transformation Efficiency

How to Find Transformation Efficiency
How to Find Transformation Efficiency
BACKGROUND: The cell is the basic unit of all living organisms, including bacteria and humans. DNA, the genetic blueprint needed to construct an organism, is present in the majority of cells. In nature, bacteria utilise transformation to exchange tiny DNA fragments.
Using the “heat shock” approach, in which charged ions and a fast change in temperature drive bacteria to absorb DNA from the surrounding environment, it is possible to coerce cells to take up DNA in the laboratory.

In practice, transformation is extremely inefficient, with only one out of every 10,000 cells successfully incorporating plasmid DNA. Since around a billion cells are employed in a transformation experiment, just a small number of cells must be converted for a positive outcome.

We can calculate the transformation efficiency using the data from our experiment to determine how well our transformation worked. This is a quantitative measure of the number of cells transformed per 1g of plasmid DNA. Essentially, it is a measure of the experiment’s success.

TO CALCULATE THE TRANSFORMATION EFFICIENCY

To calculate the efficiency of the transformation, we require the following data:

In micrograms, the amount of plasmid utilised for transformation. 10 nanograms, or 0.01 micrograms, of plasmid is typically used for transformation.
The volume of the cell suspension at the end of recovery. This comprises both the volume of competent cells and the volume of recovery broth.

The amount of cells cultured on nutritional agar. This will allow us to determine the proportion of transformed cells that were plated, since we will not often plate the complete volume of altered cells.
The quantity of colonies found on the plate. Each colony represents a single cell that has undergone transformation.

Determine the quantity of colonies on the transformation plate. Marking each colony with a lab marking pen is a practical way to keep track of the number of colonies counted.

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DETERMINE THE TRANSFORMATION RATIO USING THE FOLLOWING FORMULAS:

Electrophoresis of peptides
To determine the number of transformants per microgram of DNA, plug the values into the preceding equation.

The average transformation efficiency ranges from 1 x 104 to 1 x 108 cells per g of plasmid. Altering the heat shock conditions and analysing the results would be a fascinating way to investigate this idea in class.

Can your transformation be made more effective by including additional DNA or adjusting the time or degree of the heat shock? Determine whether it works by attempting it.

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