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Old 08-29-2016, 08:29 AM   #11
giderk
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Location: Saint Louis, MO

Join Date: Apr 2015
Posts: 12
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Sorry for the delayed response @thermophile, have been at a conference for past week. I am doing 18SrRNA sequencing and the annealing temperatures of the degenerate primers I am using ranges from 60-63.5 and 58-62.5, respectively. I haven't done any runs yet, I have just been worried that the run would fail because I didn't realize the Tm mistake before prepping the libraries. I am scheduled to run the MiSeq tomorrow though.

Although I lose a bit of diversity I decided to extend the sequencing primers into the amplicon enough to raise the Tm to an average of 65. For some specific groups of organisms that I am missing, I have designed additional sequencing primers that I am going to spike in. To test that the sequencing primers work, I am running a MiSeq Nano V2 kit. If the sequencing primer cocktails work, I'll run the MiSeq 2x300 kit later in the week.

Will share the outcome when I know what it is.
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