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I'm an undergraduate at Marshall University doing research with microalgae (specifically Chlorella). I'm attempting to transgenically modify the cells using an expression vector and have no experience in this area. As far as I understand, this will involve creating a cDNA clone of the target gene using reverse transcriptase on isolated mRNA (how is it possible to isolate a single mRNA in particular?) from the algae. Upon PCR amplification of the cDNA product, specific restriction enzymes are then used to insert the cDNA into an appropriate expression vector. The vector is then inserted into the cells, possibly by means of projectile bombardment or electroporation.
Am I oversimplifying this process? I already have the gene sequence and have identified its open reading reading frame, as well as potential primers with appropriate melting temperatures. Unfortunately, I'm unsure about what steps I should take next. Please help!
In addition, I realize microalgae are categorized as protists, despite their similarities to plants (cell wall, photosynthesis, etc.). However, for most intents and purposes in an experimental context (DNA/RNA extraction and isolation, protein extraction, etc.), can I consider them plants? If not, should I consider looking into protocols and lab manuals geared specifically toward working with microalgae?
Thanks for any advice! =]
|cdna , electroporation , microalgae , transformation , vector|
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