I did a PCR reaction, cloning and sequencing on what is supposed to be dinoflagellate mRNA which I converted to cDNA. The resulting sequence is most similar to slime mold proteins. How can I check if the slime mold protein is just most similar to the dinoflagellate protein or weather my dinoflagellate culture is contaminated with some species of aquatic slime mold? I was thinking about amplifying and sequencing with the use of 18s primers but I'm not sure if any slime mold sequence will come up if the slime mold RNA is not very abundant in my sample? It was very difficult to get the original PCR to work as well. Any help is welcome, thanks in advance!
Try to use the Nanodrop , it's software program has cDNA measurement
You can use primers specific only for mycetozoa and check is you have any trace of these species in your samples. If yes, it means you have contamination. On the other hand you can't exclude the similarity of sequences. Try to identify the sequence of "your slime mold" first. At the end maybe you'll find a new species. :)
Thank you all for your answers.
@Artur: I know I have dinoflagellate cDNA because I used specific primers to check. But I do want to know if the gene I cloned (using TOPO TA cloning) comes from a dinoflagellate or a slime mold.
@Agnieszka: I would like to use primers specific for mycetozoa. However, I'm afraid it will be a species that has not been sequenced before so I don't have any information about how conserved it's genetic code is compared to other (sequenced) slime mold species.
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