Research started!
Well, at least the preliminary work. I'm trying to analyse G-protein coupled receptor heterodimerisation [1] via reconstitution of two (enhanced) yellow fluorescent protein fragments. This means that I'll have to construct plasmids for fusion proteins of the appropriate receptors with either the N- or C-terminal part of YFP. In close proximity due to receptor interaction, the two YFP halves should re-associate to form a functional fluorophore. Right now I'm busy with primer design and which restriction enzyme sites to implement. Sounds lovely, doesn't it?
[1] to be precise: neuronal nucleotide and purinergic receptors.
Labels: final year research
2 Comments:
Something I've always wondered: How do you go about picking a primer sequence??
Well, you need one forward and one reverse primer, each of which having a 3' hydroxy group the polymerase can prime DNA synthesis from. This means that the forward primer constitutes a partial copy of the coding strand 5' region and it would anneal to the non-coding strand 3' region, while the reverse primer is a partial copy of the non-coding strand 5' region and anneals to the coding strand 3' region.
The cool thing is that you could implement whatever sequences you like in the overhangs that don't anneal to your template DNA, as long as your complementary region is long enough to ensure specificity of base-paring (20nt are sufficient). Even point-mutations are quite easily introduced by correct primer design.
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