Fluorescence Microscopy, Cricket, stART, & Gene Specific Primers

I uploaded a new video to YouTube this weekend (posted below) chronicling my latest efforts in lab and a few things on life around Clark.

This video touches on:

• how I'm trying to use fluorescence microscopy to determine which cultures of my transformed diatoms possess our experimental plasmids that express GFP
• the Clark international students playing cricket on campus
• the annual Worcester art in the street festival (stART), held along Park Avenue

Since this video was recorded, I've made further steps in my project. In order to test cultures of diatoms sooner for their presence of GFP and at a much higher volume, I've been designing gene specific primers to amplify regions of DNA in the diatoms, that would yield bands of DNA if the GFP is present. There are four main sets of primers I've been designing and will use in PCR experiments:

By using the standard NR-eGFP-NR construct as an example, the sets of primers would like the above picture, where orange highlights show where primers would amplify if the sequence exists in the DNA. Primer (1) would yield segment [a]: a portion of the 5' UTR, eGFP (enhanced GFP), and a portion of the 3' UTR. Primer (2) would yield segment [b], a portion of the 5' UTR and a portion of eGFP; primer (3) would yield a segment [c], portion of eGFP and a portion of the 3' UTR. Primer pairs to amplify the (1, 2, & 3) regions will be made for all of the gene constructs, that is NR-eGFP-NR, NR-eGFP-Actin, NiR-eGFP-NiR, and NiR-eGFP-Actin. Therefore only a specific set of primers will be used per diatom culture, depending on which plasmid it is supposed to have.

All of the plasmid however will be amplified with the fourth and final primer pair. Primer (4) would yield an eGFP segment [d] if the diatom cells had it. This will be the most important primer result out of all of them.

Once we have these primers at hand, I can screen many colonies at once. All I need to do is take a small DNA sample from a colony, add it to a PCR reaction, and run the reaction. I should be able to quickly decipher which colonies have GFP based on an electrophoretic gel.