EnzymoPure™ pA-Tn5 Transposase is an E.coli-derived fusion protein of mutated Tn5 transposase and protein A, with high Tn5 transposase activity. The pA-Tn5 Transposome comprising the pA-Tn5 transposase and appropriate sequencing adapters can be used for CUT&Tag assays.CUT&Tag (Cleavage Under Targets and Tagmentation) is an emerging approach for protein-DNA interaction studies with the advantages of high specificity, low background noise, high sensitivity, good reproducibility and low cost. It has been used as a substitute of the traditional ChIP-Seq (Chromatin Immunoprecipitation-sequencing) in protein-DNA interaction studies. The CUT&Tag technique utilizes the ability of Protein A or Protein G on the pA/G-Tn5 Transposome to specifically bind antibodies of the target protein, enabling the Tn5 transposome to bind the target protein. Subsequently, Mg2+ is added to activate Tn5 Transposase which cleaves the target protein bound DNA into fragments and simultaneously attaches the sequencing adapters to both ends of the DNA fragments. After PCR amplification with Dual-indexed PCR primers, the DNA library is ready for Next Generation Sequencing (NGS) [1].EnzymoPure™ pA-Tn5 Transposase is a recombinant protein expressed in E.coli and purified by Aladdin's proprietary PerfectProtein™ Technology Platform. Compared with wild-type Tn5 transposase, this product has at least 1000-fold higher insertion efficiency, with good transposition randomness, high stability and easy identification of the insertion site.Enzyme storage solution
Precautions：
This product contains 50% glycerol and will not freeze when stored at -20ºC. Avoid storing at -80ºC as it will freeze and repeated freeze-thaw may reduce the enzymatic activity of the product. This product is viscous. Be careful to take the correct amount when aspirating and avoid the formation of air bubbles when mixing it with samples.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use：
Construction of the pA-Tn5 transposon required for CUT&Tag experiments.1.Sequence of MEs and Adapters.ME: 5'-[phos]CTGTCTCTTATACACATCT-3'Primer-1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'Primer-2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'Note: This adapter sequence is referenced to the Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030) and can also be adjusted to meet the requirements of different high-throughput sequencing platform.2.Preparation of Adapter 1 and Adapter 2.Set up the following annealing reactions for Adapter 1 and Adapter 2, respectively. The final concentration of Adapter 1 or Adapter 2 in the reaction is 200μM. Component Volume Annealing Buffer for DNA Oligos (5X) 10μl ME (500μM) 20μl Primer-1 or Primer-2 (500μM) 20μl Total Reaction Volume 50μl Transfer the reactions onto a thermal cycler and run the thermocycling conditions as below: Step Temperature 1 95ºC, 2min 2 95ºC, 8s, -0.1ºC per cycle 3 GOTO step 2700 cycles 4 4ºC forever 3.Preparation of the pA-Tn5 transposome.Mix pA-Tn5 Transposase, Adapter 1 and Adapter 2 in a molar ratio of 1:0.5:0.5 according to the table below, mix well by pipetting, and incubate for 1 hour at room temperature. Component Volume pA-Tn5 Transposase (40μM) 10μl Adaptor 1 (200μM) 1μl Adaptor 2 (200μM) 1μl Note: The amount of pA-Tn5 Transposase in the mix can be increased if required, but the concentrations of Adapter 1 and Adapter 2 must be the same. The prepared pA-Tn5 transposome can be used directly in CUT&Tag experiments or stored at -20ºC for future use.References:1.Hatice S. Nat Commun. 2019.10: 1930.Related Products: Cat. No. Product Name Pack Size D0251 Annealing Buffer for DNA Oligos (5X) 1ml D7102S Tn5 Transposase 800pmol D7102M Tn5 Transposase 4000pmol D7103S DNA Library Quick-Prep Kit (For Illumina) 20T D7103M DNA Library Quick-Prep Kit (For Illumina) 100T P2156-200μl Concanavalin A Magnetic Beads 200μl P2156-1ml Concanavalin A Magnetic Beads 1ml P2156-5ml Concanavalin A Magnetic Beads 5ml D7110S pA-Tn5 Transposase 800pmol D7110M pA-Tn5 Transposase 4000pmol D7112S pG-Tn5 Transposase 800pmol D7112M pG-Tn5 Transposase 4000pmol
