See spreadsheets - Gentoo Experiment Details 18s
Each number corresponds to each worksheet
1. Samples and Date
Gentoo penguin scats were collected from Cumberland Bay, South Georgia (from the Maiviken colony). Visits were made weekly between 3 April and 19 Sep 2018 (one visit in June was missed owing to avalanche risk). During each of the 24 visits, 25 fresh scats were collected, producing a total of 600 samples. Samples were scooped into a 2 ml plastic screw-top tube containing 80% ethanol with a clean spatula and frozen at -20 degrees.
DNA was extracted from ~30 mg of faecal material using the Promega Maxwell RSC Tissue DNA Kit.
Each extraction contained a soft part or ~500ml of EtOH slurry. The samples were spun down, the EtOH was poured off, the sample was re-suspended in 120ul of S.T.A.R buffer and homogenised. 100ul of the supernatant was added to well number 1 and samples were eluted in 100ul of TE.
2. Plate Layout
Samples were diluted 1/10 and plated out on 96 well plates. Each plate had a positive control (fish, squid, shrimp DNA mix) and a negative PCR control.
3. 1st Round PCR.
All samples were analysed using a highly conserved metazoan primer set that amplifies a region of the nuclear 18S gene ( McInnes et al. 2017a). The first round PCR is to amplify the target marker and add sample-specific (7bp) multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers. See sheet for PCR conditions.
4. 2nd Round PCR
The second round PCR is to add sequencing adapters and additional 8 bp MIDs. See sheet for PCR conditions.
5. Miseq
MiSeq genome sequencer (Illumina), using the MISEQ V2 reagent kits (300 cycles).
See sheet for sample layout, i5 and i7 adapters and first round MID tags
See spreadsheet - Gentoo Experiment Details Fish and Krill
The scat samples containing prey DNA sequences from the 18S analysis (n=222) were characterised with two other primer pairs allow species-level identification for fish and krill.
1. Samples
The samples positive for prey DNA and their plate layout
2. and 3. 1st Round PCR Krill /Degenerate
The first round PCR is to amplify the target marker and add sample-specific (6bp) multiplex-identifier (MID) tags (forward and reverse primer) and Illumina sequencing primers. See sheet for PCR conditions.
4. 2nd Round PCR
The second round PCR is to add sequencing adapters and additional 10 bp MIDs. See sheet for PCR conditions.
5. Miseq
MiSeq genome sequencer (Illumina), using the MISEQ V2 reagent kits (300 cycles).
See sheet for sample layout, R and F adapters and first round MID tags.
This work was completed as part of AAS project 4556.
