Saturday, October 15, 2011

Diatom Colony Screen

A new lab video!

In this video I discuss designing a colony screen protocol for my transformed lines of diatoms. In one of my recent posts, I posted this picture diagram which oversimplified the process:

While the PCR steps are the same (starting in the top left panel with the diatoms being combined with the PCR mix), I discovered that diatoms grown in liquid culture (i.e. their natural state) needed extra care in preparing them for a colony screen.

Let's step back a bit and quickly talk about colony screens (in case you didn't read my above Tumblr post). A colony screen is a modified PCR reaction*, which is a cyclical amplification of a short sequence of DNA, exponentially copying the targeted DNA strand. (*Even though PCR stands for polymerase chain reaction, PCR is usually said out loud in conversation as PCR reaction.) Often the DNA source for a PCR reaction is a purified, such as plasmid DNA purified by a process to separate plasmid DNA from proteins and genomic DNA.

But colony screens use a colony of cells to supply the DNA for the PCR reaction. By initially lysing the cell by cooking it at a high temperature for a period of time, the cell's DNA is released into the PCR reaction mix and then amplified in the reaction. Colony screens are used to screen cells for the presence of DNA--that is if the targeted sequence of DNA is present, it will be amplified by the reaction. If a reaction gives a positive band on a gel (bottom right of the colony screen chart), then we know that the DNA from the cells in that particular reaction also have the DNA of interest.

This colony screen method should in theory work for other cells, like diatoms. My initial trial successfully amplified GFP from diatom colonies growing on agar plates, but it didn't work for diatom cultures growing in liquid media, a distinction I didn't make at the time.

I soon realized after a failed trial of screening  diatoms solely from liquid cultures that the residual salt water from their growth media was throwing off the delicate chemistry of the PCR reaction. This graphic paired with the above video discusses just this.

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