# Pretreatment Method for DNA Barcoding to Analyze Gut Contents of Rotifers
## Materials and Methods
### DNA Analysis Procedure  
The DNA was isolated from the filter paper using DNEasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions on a clean bench (Supplementary Materials). In order to reduce potential contamination during DNA extraction and the amplification process, we minimized contamination sources by separating each sample, using bleach sterilized gloves, and instruments sterilized by autoclave and ethanol.  
A polymerase chain reaction (PCR) amplification was performed using AccuPower Hot start PCR PreMix (Bioneer, Korea) with genomic DNA and primers in a final volume of 20 \(\upmu\)L. Primers were selected for specific detection of each potential prey community for rotifers. Phytoplankton and the components of the microbial food web (bacteria and protozoa) were considered as potential food sources for rotifers [6]. At this time, since we could not find a suitable primer to detect only heterotrophic nanoflagellates (HNF) specifically, we applied a universal primer for eukaryotes to HNF [38; 39]. PCR conditions for each primer in a thermal cycler (Bio-rad, California, USA) were summarized in Table 2. PCR products were separated using 1.5% agarose gels (AccuPrep(r) PCR/Gel DNA Purification Kit (50 reactions) [K-3038]), and the appeared band from PCR products was extracted and sequenced in both directions by capillary sequencing at Bioneer Co. (Daejeon, Korea). Additionally, cloning was carried out using the pGEM-T easy vector (Promega, Madison, USA) to confirm direct sequencing results. Cloned plasmid DNA was isolated according to the alkaline-lysis method using Labopass Plasmid Miniprep kit (Cosmogenetech). Individually isolated plasmid DNA was then digested using the restriction enzyme EcoRI to confirm insertion. Positive clones for each sample were analyzed to species-specific sequences with SP6 primers using an automated 3730 DNA analyzer (Applied Biosystems, Foster City, USA).  
Sequence alignment was performed using Clustal W 2.0 [40]. A BLASTn [41] search was performed to identify sequences with the best hits. In the GenBank nucleotide collection database, the organisms, which were included in the database search, were optimized for highly similar sequences by BLASTn, and selected by high identity (%).  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Food source** & **Primer** & **Sequence (S-37)** & **Base Pair** & **Ref.** \\ \hline Chlorophyceae & ChloroF & TGG CCT ATC TITG TTG GTC TGT & & \\ (18s rRNA) & ChloroR & GAA TCA ACC TGA CAA GGC AAC & & 473 bp & \\
94 °C, 3 min - 35 cycles [94 °C, 1 min - 55 °C\({}^{*}\), 1 min - 72 °C, 1 min] - 72 °C, 1 min & & \\  & M13F- & TGT AAA ACG GCC AGT ATT CCA GCT & & \\ Diatomea & D512 for & CCA ATA GGC & & 390–410 bp & \\ (18s rRNA; V4) & M13R-D978 rev & CAG CAA ACG ACT ATG GGC ACT & & \\  & & ATG GTA TCT AAT C & & \\  & 94 °C, 2 min -55 cycles [94 °C, 45 - 53°C\({}^{*}\), 53°C\({}^{*}\), 45s - 72 °C, 1 min] & & \\  & \(>\)35 cycles [94 °C, 45 - 53°C\({}^{*}\), 45s - 72 °C, 1 min] - 72 °C, 10 min & & \\ \hline \hline \end{tabular}
\end{table}
Table 2: PCR primers used for detecting rotifer food sources.