Hello, I want to determine the photosynthetic microbial population in some wastewater samples through a genetical analysis. I have a cell counter that can hopefully help me to distinguish between different species, but how can I separate the genetic material from each to perform my PCR and the following sequencing? I was thinking to use DGGE, but what do you suggest?
Hi Lorenza,
I don't know how time and funding you can spend on this and I don't have any experience with DGGE, but I would always recommend to isolate single species from the raw material. This will make it easy to isolate DNA and use eukaryote specific primers to amplify rDNA marker regions typically used for micro algal strains (18S - small subunit, 28S small subunit, ITS or chloroplast genes, i.e. rbcL, etc.). This way you can also do morphological identification which is always useful, especially since reference sequences aren't available for all known and unknown species and because the DNA seldom tells the whole story. This is a relatively cheap way to do it, but there are some downsides. It's very time consuming, and you probably won't be able to get all the present species in culture.
If you only want to do molecular identification, cloning or next gen sequences (454 pyrosequences, Illumina sequencing, etc.) of the whole wastewater sample is a way to do it. This is less time consuming but way more expensive. Also it demands higher grade of processing and filtering of the dataset.
Hope this helps!
Anders Wold
Thank you Anders,
yes, this is the point, the isolation through conventional methods, basically plating on solid media after serial dilutions, it's really time consuming and I have the experience that some of the species are very hard to isolate. Anyway, I just want to know the population in the samples, because we are planning to follow it through the year and see how it is influenced by the environmental conditions (light, temperature, ecc.). That's why I was looking for something faster and I think DNA discrimination is the best way to identify the species. But I don't know how to separate DNAs before my amplification!
Thank you so much Tim, your answer was really really helpful! I saw that the chair of the National platform steering board for NGI is from my University, great, I will ask him.
Please see the papers for your reference
https://www.researchgate.net/publication/327381747_Microalgae_Cultivation_Using_Various_Sources_of_Organic_Substrate_for_High_Lipid_Content
Article Evaluation of Lipid Content in Microalgae Biomass Using Palm...
//www.researchgate.net/publication/316675390_Evaluation_of_Lemna_minor_and_Chlamydomonas_to_treat_palm_oil_mill_effluent_and_fertilizer_production
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