Transformation is the process of taking DNA, typically plasmid DNA, and inserting it into competent bacterial cells. This allows scientists to change the bacteria to reflect desired changes, like fluorescent or chromogenic proteins. Like all experiments, transformations can be unsuccessful sometimes, so it is important to follow all protocol instructions as closely as possible.
We recently did some experiments to “mess up” some transformations. Ultimately, we found that when the source plate is old, the colonies aren’t isolated well enough, and they aren’t selected properly, this is the main cause of a transformation failure.
The reason source plates are so important is because it is where the bacterial colonies come from that will be transformed with the exogenous DNA. These colonies need to be able to be made “competent” which means that their cell walls will be able to open up to welcome this DNA in! Most commonly, cells are made competent with ice-cold calcium chloride (CaCl2). The chemical ions of CaCl2 allow the DNA to attach to the outside of the bacterial cell, and the heat shock steps allow the membrane to open up to let the DNA into the bacterial cell. Once the DNA has entered the bacterial cell, it is considered transformed! Selection and screening assays can be done to isolate these transformed bacteria. Most commonly this is done by adding an antibiotic and/or promoter to the agar so that the bacteria containing the plasmid will grow. For example, in our GFP kits, the transformed bacteria will grow on agar that contains ampicillin and IPTG.
When you’re selecting colonies, you want colonies that are well isolated, uniform in color and shape, and roughly 1-1.5 mm in diameter. These colonies also tend to be a cream color and look like they are sticky. When the colonies on the source plate sit for longer than 24 hours, the transformation efficiency of the experiment greatly decreases. The colonies and the agar they are on begin to dry up, are harder to select off of the plate, and are also harder to re-suspend and make competent in the CaCl2. Which will cause a trickle down effect and the transformation will not be successful if the cells can not take up the plasmid DNA.
Of course, making sure that you follow timing and temperature requirements in your transformation experiment is just as important! All of this is always in detail in the experimental protocol of any EdvoKits you may be using!
Check out this YouTube video for more detailed information on how to streak your source plates for the best transformation results! Also, check out this free, printable informational sheet about streaking plates: https://www.edvotek.com/site/pdf/Transformation_Source_Plates.pdf
Edvotek Transformation Experiments:
Transformation of E. coli with GFP
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