Purified ChIP DNA from a standard ChIP for H3K4me3 on PBMCs was measured using a Qubit fluorometer (Life Technologies) and then diluted in 10mM Tris-Cl pH 8.5 supplemented with 0.1% Tween-20 to 100 pg, 10 pg, or 2 pg total DNA. The tagmentation reaction was performed for 5 minutes at 55°C in a 10 μl reaction containing diluted DNA, 5 μl 2x tagmentation buffer (Illumina) and 1 μl (for 100 pg DNA) or 0.5 μl (for 10 pg and 2 pg) 1:10 diluted Nextera Tag DNA Enzyme (diluted in precooled TE/50% glycerol). The tagmented DNA was amplified with the Nextera DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with the following program: 72°C 5 min, 98°C 30 s, 14 cycles of 98°C 10 s 63°C 30 s 72°C 30 s, and a final elongation at 72°C for 1 min. Libraries were purified using SPRI AMPure XP beads with a beads-to-sample ratio of 1.5:1. Purified ChIP DNA or deproteinized input DNA from K562 ChIP was prepared as for PBMCs with slight modifications: 5 ng of ChIP DNA was taken for the tagmentation reaction using 0.5 μl of a 1:10 diluted Tn5 transposase in a 5 μl reaction at 55°C for 5 minutes. DNA was purified with the MinElute kit (Qiagen) and amplified with the Kapa HiFi HotStart ReadyMix (Kapa Biosystems).
Tagmentation buffer
Manufactured by Illumina
The 2x tagmentation buffer is a laboratory reagent used in DNA library preparation workflows. It serves as a core component in the tagmentation process, which is a key step in generating DNA libraries for sequencing applications. The buffer facilitates the fragmentation and tagging of DNA samples, enabling subsequent steps in the library preparation protocol. This product provides the necessary chemical environment for the tagmentation reaction to occur effectively.
Automatically generated - may contain errors
2 protocols using tagmentation buffer
1
Tagmentation and Library Preparation for ChIP-seq
2
Tagmentation and Library Preparation for ChIP-seq
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Purified ChIP DNA from a standard ChIP for H3K4me3 on PBMCs was measured using a Qubit fluorometer (Life Technologies) and then diluted in 10mM Tris-Cl pH 8.5 supplemented with 0.1% Tween-20 to 100 pg, 10 pg, or 2 pg total DNA. The tagmentation reaction was performed for 5 minutes at 55°C in a 10 μl reaction containing diluted DNA, 5 μl 2x tagmentation buffer (Illumina) and 1 μl (for 100 pg DNA) or 0.5 μl (for 10 pg and 2 pg) 1:10 diluted Nextera Tag DNA Enzyme (diluted in precooled TE/50% glycerol). The tagmented DNA was amplified with the Nextera DNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions with the following program: 72°C 5 min, 98°C 30 s, 14 cycles of 98°C 10 s 63°C 30 s 72°C 30 s, and a final elongation at 72°C for 1 min. Libraries were purified using SPRI AMPure XP beads with a beads-to-sample ratio of 1.5:1. Purified ChIP DNA or deproteinized input DNA from K562 ChIP was prepared as for PBMCs with slight modifications: 5 ng of ChIP DNA was taken for the tagmentation reaction using 0.5 μl of a 1:10 diluted Tn5 transposase in a 5 μl reaction at 55°C for 5 minutes. DNA was purified with the MinElute kit (Qiagen) and amplified with the Kapa HiFi HotStart ReadyMix (Kapa Biosystems).
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