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Old 08-15-2017, 08:54 AM   #2
adam.geber
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Location: United States

Join Date: May 2014
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0.8x cleanups shouldn't be able to touch your 550bp amplicons. Are you using the same polymerase recommended by Illumina? If not, then you'll likely have to do some further optimization via gradient PCR to confirm the ideal conditions -- see this thread for some starting suggestions. Also, are you using a positive control in your amplifications? I don't know much about sludge bacteria but you need to rule out issues of limited starting template or inefficient DNA extraction from your samples. Zymo makes a very good microbial DNA standard that you can use for both these purposes but if you don't want to spend the money and you don't need a standard with known species you can always just use an oral swab as input for your DNA extraction.
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