Tn5 Transposase Activation and DNA Fragmentation

Tn5 reactions were assembled by mixing 14 μL H₂O, 4 μL 5× TAPS-MgCl₂-PEG 8000 or 5× TAPS-DMF, 1 μL target DNA at 50 ng/μL, 1 μL of the Tn5, preassembled with A/B-MEDS oligonucleotides at 12.5 mM. Buffers used were as follows: 5× TAPS-PEG 8000 (50 mM TAPS-NaOH at pH 8.5 [RT], 25 mM MgCl₂, 40% PEG 8000) or 5× TAPS-DMF (50 mM TAPS-NaOH at pH 8.5 [RT], 25 mM MgCl₂, 50% DMF). For library production, the reactions were incubated for 7 min at 55°C, and then 5 μL 0.2% SDS (final 0.02%) was added and Tn5 was inactivated for 7 min at room temperature or 55°C. Typically 5 μL of the Tn5 reactions was used directly in the subsequent enrichment PCR amplification of libraries for the Illumina sequencers. Successful libraries were also generated using only the index adaptors as they contain the entire sequence of the PCR primers (FC-121-1012 and FC-121-1011).
For testing transposase activity, reactions were set up as above, with 50 ng HMW DNA as substrate. To stop the reactions, 0.5 μL proteinase K (20 mg/mL; Qiagen) was added to each reaction, followed by incubation for 7 min at 55°C. The samples were then analyzed by agarose gel electrophoresis. Typically all of the HMW DNA was converted to fragments of average size 400–500 bp. An image of a typical activity assay is shown in Figure 1C.