Versatility of the BOMB protocols for nucleic acid isolation.

Nucleic acid extraction from various sample origins using the BOMB extraction protocols (S1 Appendix, BOMB protocols 4–8). (A) Size exclusion of GeneRuler DNA Ladder Mix (Thermo) using BOMB silica-coated magnetic beads (S1 Appendix, 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 (Gallus gallus domesticus) gDNA. The two rightmost lanes contain the gel extracted bands from input lane using S1 Appendix, 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 Escherichia coli cells. The lanes labelled “gDNA” contain gDNA isolated from untransformed cells, whereas lanes denoted as “gDNA+plasmid” contain the total DNA extracted from E. coli carrying pHAGE-EFS plasmid (S1 Appendix, BOMB protocol 7.1). Purified pHAGE/EFS plasmid is shown in the “plasmid” lane (S1 Appendix, BOMB protocol 5.1). MW: GeneRuler DNA Ladder Mix (Thermo). (D) Total plasmid DNA yield (μg) extracted from E. coli, 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 S1 Appendix, BOMB protocol 5.1; red dots represent samples processed using a commercial kit. (E) TNA isolation (S1 Appendix, BOMB protocol 6.6) from E. coli (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 S1 Appendix, 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 S1 Appendix, BOMB protocol 7.1. (H) Total RNA isolated from 500,000 HEK293 cells following S1 Appendix, 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 (Oryctolagus cuniculus) tissues using BOMB silica-coated magnetic beads following S1 Appendix, BOMB protocol 6.3. MW: Hyperladder I (Bioline). (M) TNA isolation from yeast (Saccharomyces cerevisae) using S1 Appendix, BOMB protocol 6.5. MW: GeneRuler DNA Ladder Mix (Thermo). (N) TNA isolation from clover (Trifolium repens), daisy (Bellis perennis), and ryegrass (Lolium perenne) according to S1 Appendix, BOMB protocol 6.4 with carboxyl-coated magnetic beads. MW: Hyperladder I (Bioline). (O) Total DNA yield (ng) of representative extractions from leaves of T. repens, plotted against the A260 nm/A280 nm ratio of each sample. (P) TNA isolation from 50 ml of lake water following S1 Appendix, BOMB protocol 6.7. MW: GeneRuler DNA Ladder Mix (Thermo). Underlying data for Fig. 2 can be found in S1 Data. 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.