| Summary: | Progress Code: completed Statement: The values provided in temporal coverage are approximate only and correspond to rough dates for the project as a whole. 1st Experiment 24/11/16 ************************************************************************************************ See 2016_11_24_Miseq_Sheet 1. Sanger Sequencing Plate #4 - 25mg of Tissue was extracted by AGRF. DNA was diluted to 5ng/ul. Samples were sanger sequenced with 16SAR (Palumbi) primer. If they failed, I used COI3 cocktail (Ivanova). FASTA sequences from Plate 4 are in the folder named Sanger Sequence FASTA Plate #4. Naming - Plate position, primer, sample ID. ie reater than A1-16S-AR_1952. 2. DNA and Tissue Pools of Plate 4 We wanted to explore the possibility of using a metabarcoding approach. For metabarcoding we re-examined specimens already identified from sanger sequences. We mixed DNA from many samples (n=16 or n=96) and did a single amplification (i.e. up to 96 DNA extractions processed in a single-tube marker amplification). We also took it a step further and tried blending a set amount of tissue from many fish specimens (n=16 or n=96) and did a single DNA extraction on the tissue mixes (i.e. a single DNA extraction and single tube amplification for up to 96 samples). See 2016_11_24_Miseq_Sheet for DNA and Tissue Pool mixes. 3. Miseq Run 16 samples were ran on a 250bp pe read. Each sample was amplified with 3 primer sets - COI (please note one dual labelled set was used), 12s and 16s (Primers listed on 2016_11_24_Miseq_Sheet). They were diluted 1:10 and illumina sequencing adaptors were added (please note I used same I7 and I5 per sample, so they had to be sorted on amplicon). 2016_11_24_fastq_files has the data from miseq. and 2016_11_24_merged_fastq_files has the merged files. For some unknown reason 16s tissue produced no data. 2nd Experiment 04/07/17 ************************************************************************************************* 1. DNA Extractions Plate #1, 2 and 3 - 25mg of Tisse was extracted by AGRF. DNA ...
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