| Product Description | Aladdin's EnzymoPure™ Tn5 Transposase is a hyperactive, mutated form of Tn5 Transposase and a highly efficient enzyme for insertion of a Tn5 transposon into both eukaryotic and prokaryotic DNA. This product specifically recognizes DNA fragments containing Mosaic End (ME) sequences at each of its ends (including primers containing ME sequences), resulting in the formation of a Tn5 transposome which randomly attacks and cleaves the target DNA to insert the DNA fragment it carries.EnzymoPure™ Tn5 Transposase is recombinantly expressed in E.coli and purified on Aladdin's PerfectProtein™ technology platform. It possesses good randomness, high stability, and a transposition frequency that is 1000-fold greater than wild type Tn5 transposase.Figure 1. Schematic diagram of transposition reaction catalyzed by Aladdin's EnzymoPure™ Tn5 Transposase .EnzymoPure™ Tn5 Transposase catalyzes a multi-step “cut and paste” transposition reaction (Figure 1). Initially, the enzyme binds the ME sequence of the transposon to form a complex①. Then two Tn5 transposases at two ends of one transposon associate and circularize the transposon through C-terminal interactions, forming a Transposase dimer complex②. In the presence of Mg2+, the Tn5 transposase cleaves the donor DNA and carries the donor DNA fragment to form the Transposome③. The Tn5 transposome recognizes and binds the target DNA to form a complex of transposome and target sequence④. In the presence of Mg2+, the Tn5 transposase cuts the target sequence and insert the donor DNA fragment it carries into the target sequence, forming the transposed DNA⑤. Finally, the 9bp sticky ends created by transposition can be blunted by DNA polymerase and ligase, forming a 9bp sequence duplication immediately flanking the transposon insertion site.s
Application： Fragmentation and tagmentation during next-generation sequencing (NGS) library preparation; introduction of sequencing primers into cloned DNA or plasmids; construction of bacterial gene knockout libraries; engineering of novel bacterial strains; insertional inactivation of target genes; insertion of T7 promoter and resistance marker into target DNA.Storage Buffer: 50mM HEPES (pH7.2), 100mM NaCl, 0.1mM EDTA, 1mM DTT, 0.1% Triton X-100, 50% (v/v) Glycerol.Reaction Buffer (5X): 50mM HEPES (pH7.2), 500mM NaCl, 50mM MgCl2.Stop Buffer (5X): 50mM EDTA (pH8.0).For in vitro transposition, use 1μl of Tn5 Transposase (40μM) per reaction. D7102S and D7102M are sufficient for 20 and 100 reactions, respectively.
Precautions： This product contains 50% glycerol and does not freeze at -20ºC. Store this product at -20ºC, but not -80ºC to avoid repeated freeze-thaws.This product is viscous, so be careful to take the right amount when aspirating. After adding to samples, mix well thoroughly by pipetting and avoid the formation of air bubbles. Tn5 Transposase uses DNA as substrate, but not RNA.This product is only applicable to bacteria for transposon insertion. Eukaryotic cells are difficult for the insertion of transposon due to the blockage by nuclear membrane. Please optimize the reaction conditions according to the relevant literature.For bacteria related studies, please do not use chemically competent cells. It is recommended to use electrocompetent cells with the electrotransformation efficiency higher than 106cfu/µg.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： 1. In vitro transposon insertiona. Generation of Tn5 transposon. The Tn5 transposon is a DNA fragment consisting of target DNA sequence flanked by the 19bp ME sequence at both ends, and can be synthesized by PCR using primers that have non-homologous tails with 19-base ME sequnces at 5’-ends and homologous sequences to the template at 3’-ends. For optimal transposition efficiency, primers needs to be 5' phosphated. Please refer to Figure 2 for a typical Tn5 transposon.ME sequence: 5'-[phos]CTGTCTCTTATACACATCT-3'Figure 2. Schematic diagram of Tn5 transposon. The ME sequences at both ends is uniquely and specifically recognized by NGSTM T5 transposase (, D7102)b. Prepare the reaction mix as follows:ComponentQuantityReaction Buffer (5X)2μlTarget DNA*0.2μgEquivalent Moles of Tn5 Transposonx μl Tn5 Transposase (40μM)1μlddH2Oo 10μl*Note: Calculate the moles of target DNA in the reaction and add equivalent moles of Tn5 Transposon to avoid multiple insertions. Target DNA (μmol) = Target DNA (μg)/[(base pairs in target DNA)×660]. For example, the moles of 0.2μg 5000bp target DNA is 0.2μg/ [5000bp×660]=0.06×10-6μmol = 0.06pmol.c. Mix well, incubate at 37ºC for 2 hours.d. Add 2μl of Stop Buffer (5X), mix well and incubate at 55ºC for 10 minutes.e. Electroporate competent cells with 1μl of reaction mix and plate on selective media. The remaining of reaction mix can be stored at -20ºC. The recommended electroporation conditions: 50μl competent cells, 1μl reaction mix, 2mm electroporation cuvette, 2500V, and 5ms. The electroporation conditions can be optimized based on the result. The number of transposed clones depends mainly on the endogenous restriction modification system, competent cell type, and the transformation efficiency of competent cells. 2. Production of Tn5 transposome and in vivo insertiona. Prepare the reaction mix by adding reagents in the following order:ComponentQuantityTn5 Transposon DNA (100μg/ml in TE Buffer)2μl Tn5 Transposase (40μM)4μlGlycerol (100%)2μlTotal Reaction Volume8μlNote 1: This reaction does not need the presence of Mg2+. Do not use the Reaction Buffer (5X).Note 2: Design the Tn5 Transposon according to 1a. A custom Tn5 Transposon consisting of selective marker, a pair of recognition sequences (e.g., ME sequences) and a double-stranded target DNA fragment can be generated by PCR. Note 3: This reaction can be scaled up or down as needed.b. Mix well, incubate at room temperature for 0.5-1h.c. Electroporate competent cells with 1μl of reaction mix and plate on selective media. The recommended electroporation conditions: 50μl competent cells, 1μl reaction mix, 2mm electroporation cuvette, 2500V, and 5ms. The electroporation conditions can be optimized based on the result. The number of transposed clones depends mainly on the endogenous restriction modification system, competent cell type, and the transformation efficiency of competent cells.d. The generated transposome can be stored at -20ºC for up to 1 year.3. Preparation of Tn5 Transposome for NGS a. Sequences of ME and AdaptersME: 5'-[phos]CTGTCTCTTATACACATCT-3'Primer 1: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'Primer 2: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'Note: The above adaptor sequences are for Nextera® DNA Sample Preparation Kit (Illumina, FC-121-1030). The sequences can also be designed to meet other requirements. b. Preparation of Adapter 1 and Adapter 2Set up the annealing reaction for Adapter 1 and Adapter 2. The final concentration of Adapters after annealing should be 200μM.ComponentQuantityAnnealing Buffer for DNA Oligos (5X) 10μlME (500μM)20μlPrimer-1 or 2 (500μM)20μlTotal Reaction Volume50μlTransfer the reaction into a thermal cycler and run the following annealing conditions:StepTemperature195ºC, 2min295ºC, 8s, -0.1ºC per cycle3GOTO step 2, 700 cycles44ºC foreverc. Production of Tn5 TransposomeAccording to the table below, mix Tn5 transposase, Adapter 1, and Adapter 2 at a molar ratio of 1:0.5:0.5. Mix well by pipetting and incubate at room temperature for 1 hour. The Tn5 transposome can be used directly for DNA tagmentation, or can be stored at -20ºC. Note: The ratio of Tn5 transposase can be increased appropriately according to the experimental requirements, but the concentration of Adapter 1 and Adapter 2 should be the same. ComponentQuantity Tn5 Transposase 10μlAdapter 1 (200μM)1μlAdapter 2 (200μM)1μld. Test of Tagmentation Effect Set up the reaction according to the table below, mix gently by pipetting, and incubate at 55ºC for 5-10 minutes. Stop the reaction by adding 5μl of Stop Buffer (5X) and incubating at 55ºC for 5 minutes. The fragmentation products can be examined or purified for library preparation, as shown in Figure 3. The amount of transposome can be adjusted according to the size of fragments. Increase the amount of transposome if the fragment is too long, and decrease the amount of transposome if the fragment is too short. ComponentQuantityDNA50-100ngTn5 Transposome0.5-2μlReaction Buffer (5X)4μlddH2OTo 20μlFigure 3. Lambda DNA fragmentation by the Tn5 transposome produced by Tn5 Transposase . Reactions of 20μl containing 100ng Lamda DNA and different amounts of Tn5 transposome as shown in the figure were incubated at 55ºC for 10 minutes, followed by the addition of Stop Buffer and incubation at 55ºC for 5 minutes to terminate the reaction. The reaction products were mixed with DNA Loading Buffer (6X) (, D0071) and separated on Agarose Precast Gel . This figure is for reference only, which may vary due to different experimental conditions.
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