1. I tried to isolate diatoms by serial dilutions, and did DNA extraction and PCR. using some primers of chloroplast coding genes (tufa, rbcl,). I have single cells in each tube but they are not axenic. I had a lot of troubleshooting and the sequencing didn't work at all.
2. I tried to sort them by flow cytometry and by forward scattering, I noticed that I have another population with my samples of interest. This population is mainly small flagellates like picoplanktons, that appear as dots under the microscope. I am sorting them but the flagellates are always with my cells. I never can get rid of them at 100%. They are photosynthetic as they have absorbance at 670 nm. This is I think the main source for not geting a result in sequencing.
Could you please guide me by some advice for the success of sequencing.
Before move into sequencing reactions, how your PCR results are presented?
Hi Ines,
I have encountered similar difficulty with sequencing Chlorophyta.
Two ideas come to mind:
1) Have you tried (or do you have the ability to) start cultures with single cells picked under a microscope?
2) Could you try to use antibiotics in the growth media to eliminate eubacteria?
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