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Full details of the protocol: Library preparation protocol for 16s or ITS
3 ul of 0.01 pg/ul cross contamination oligos and 0.03 pg/ul synthgene added to 15 ul of each sample.
Concentration measured via absorption and normalized to 10 ng/ul via automated liquid handler.
Two stage PCR performed with MagBead Cleanups in between. Duplicate stage 1 PCR performed for each sample DNA and loci. First Stage Primers amplify regions of interest, add unique pairs of sequence barcoding, and a portion of the Illumina adaptor. Second stage completes Illumina adaptor addition.
After second magbead cleanup, amplicon product concentrations were measured via absorption. Products were relatively similar concentrations, with an order of magnitude. So, 2 ul of each sample was combined across 8 tubes. The 8 tubes were vortexed and spun before 50 ul of each was combined for the pool.
Finally, most amplifications showing low concentration via final absorption were run singly at a 1:500 dilution on qPCR along with the diluted full pool in quadruplicate. 2 out of the 64 individual samples did not show amplification.
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