I am studying the active fungi in marine sponges. According to this paper https://www.researchgate.net/publication/229087468_Nuclear_ribosomal_internal_transcribed_spacer_%28ITS%29_region_as_a_universal_DNA_barcode_marker_for_Fungi,
I chose the LSU as a molecular marker. However, I couldn't generate sensible products. The main band was around 1kb, which was not in a good length for 454 pyrosequencing, while the nonspecific band was about 150bp. The negative control showed there is no contamination in the PCR system. The templates I used were the ss cDNA of marine sponges. The annealing temperature was 48°C as showed in that paper. I haven't done any PCR for barcoding fungal diversity. Can anybody tell me what to do?
Article Nuclear ribosomal internal transcribed spacer (ITS) region a...
I have no idea about the time, money, facilities etc. that you have. One thing you could do is to sequence the 28S region in two steps, using two different sets of primers, one sequencing the first half of the marker, and the other one sequencing the other half. Depending on your goals, you may even only need one of the two parts.
You could sequence the 28S using a different method, such as sanger sequencing, but of course if you do environmental sequencing, that doesnt work.
You could also sequence a different marker, such as ITS (ITS1+5.8S+ITS2) which is shorter (500-700bp) and more variable. But it depends on what marker is the most sequenced for your group of fungi, if everyone sequence 28S you should definitely go for 28S.
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