No replies to this topic

[Greatman]

I faced a big experiment problem these days. I used  Crenarchaeal 16S rRNA primers(A109f/1492r ) for clone and sequencing. But most of the datas were not crenarchaeal at all. For example only two of 48 samples were crenarchaeal.(NCBI Blast) And then I reextracted the plasmid DNA and used A109f/1492r , Cren771/957r to check the plasmid DNA. I thought that would be fine. But the results letted me down again. Only 2 of 20 samples were crenarchaeal,others are bacterias. I checked the sequencing datas and found that the first run primers A109f/1492r could be found, but the second run primers Cren771/957r didn't. Why?
I had already used the second run primers Cren771/957r to check the plasmid DNA. The agrose gel confirmed the plasmids were right.
The plasmid DNA extraction was followed the mniprep procedure. I prepared the solution I,II and III.
I asked someone ,they said maybe the clones were contaminated. why?  The AOB was also followed the same condition, but most of the datas were correct.