Versatility of the BOMB protocols for nucleic acid isolation.

<p>Nucleic acid extraction from various sample origins using the BOMB extraction protocols (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocols 4–8). (A) Size exclusion of GeneRuler DNA Ladder Mix (Thermo) using BOMB silica-coated magnetic beads (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 4.1). Different volumes of binding buffer compared to sample volume were used to achieve size exclusion. Two volumes of cBB were used as a control relative to input. (B) Gel extraction of CHD1 PCR products amplified from female chicken (<i>Gallus gallus domesticus</i>) gDNA. The two rightmost lanes contain the gel extracted bands from input lane using <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 4.3 with carboxyl-coated magnetic beads. The volumes loaded are proportional (i.e., the right-hand 2 lanes represent the efficiency of capture from the left-hand lane). MW: Hyperladder IV (Bioline). (C) gDNA isolated from TOP10 <i>Escherichia coli</i> cells. The lanes labelled “gDNA” contain gDNA isolated from untransformed cells, whereas lanes denoted as “gDNA+plasmid” contain the total DNA extracted from <i>E</i>. <i>coli</i> carrying pHAGE-EFS plasmid (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 7.1). Purified pHAGE/EFS plasmid is shown in the “plasmid” lane (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 5.1). MW: GeneRuler DNA Ladder Mix (Thermo). (D) Total plasmid DNA yield (μg) extracted from <i>E</i>. <i>coli</i>, plotted against the A260 nm/A280 nm ratio for each sample. DNA concentration and purity were measured with UV-Spectroscopy (NanoDrop). Black dots represent samples extracted using the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 5.1; red dots represent samples processed using a commercial kit. (E) TNA isolation (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.6) from <i>E</i>. <i>coli</i> (left two lanes) followed by DNase I digest (right two lanes). MW: GeneRuler DNA Ladder Mix (Thermo). (F) Isolation of TNA from HEK293 cells using the <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.1 (lanes 6 and 7) followed by digestion with either DNase I (lanes 2 and 3) or RNase A (lanes 4 and 5). (G) gDNA isolated from 500,000 HEK293 cells using <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 7.1. (H) Total RNA isolated from 500,000 HEK293 cells following <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 8.1. (I) Total DNA yield (μg) of representative extractions from HEK293 cells, plotted against the A260 nm/A280 nm ratio for each sample. (J) Total RNA yield (μg) of representative extractions from HEK293 cells, plotted against the A260 nm/A280 nm ratio for each sample. (K) qPCR amplification curve of a 10-fold serial dilution (black: undiluted; dark grey: 1:10 diluted; light grey: 1:100 diluted) of RNA from HEK293 cells reverse-transcribed into cDNA. (L) gDNA isolated from various rabbit (<i>Oryctolagus cuniculus</i>) tissues using BOMB silica-coated magnetic beads following <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.3. MW: Hyperladder I (Bioline). (M) TNA isolation from yeast (<i>Saccharomyces cerevisae</i>) using <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.5. MW: GeneRuler DNA Ladder Mix (Thermo). (N) TNA isolation from clover (<i>Trifolium repens</i>), daisy (<i>Bellis perennis</i>), and ryegrass (<i>Lolium perenne</i>) according to <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.4 with carboxyl-coated magnetic beads. MW: Hyperladder I (Bioline). (O) Total DNA yield (ng) of representative extractions from leaves of <i>T</i>. <i>repens</i>, plotted against the A260 nm/A280 nm ratio of each sample. (P) TNA isolation from 50 ml of lake water following <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s011" target="_blank">S1 Appendix</a>, BOMB protocol 6.7. MW: GeneRuler DNA Ladder Mix (Thermo). Underlying data for Fig. 2 can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.3000107#pbio.3000107.s007" target="_blank">S1 Data</a>. BOMB, Bio-On-Magnetic-Beads; cBB, commercial binding buffer; CHD1, Chromodomain Helicase DNA Binding Protein 1; eTNA, environmental total nucleic acid; gDNA, genomic DNA;qPCR, quantitative polymerase chain reaction; TNA, total nucleic acid.</p>