Hieff^TM Fast Tagmentase is a mutant form of wild-type Tn5 transposase. It recognizes the inside end (IE), outside end (OE), and mosaic end (ME) sequences of the Tn5 transposon. Among these, the ME sequence exhibits the highest transposition efficiency. This product enables the efficient and random insertion of Tn5 transposons into target sequences, and is widely applied in fields such as in vitro transgenesis and next-generation sequencing (NGS) library preparation.

Specifications

Components

Storage

Store Component A (Hieff^TM Fast Tagmentase) at -85°C to -65°C. Upon receipt, complete adapter assembly within one month; the assembled product should be stored at -25°C to -15°C. Store all other components at -25°C to -15°C. The shelf life is one year.

Instructions

Taking high-throughput sequencing library preparation as an example, please read the instructions carefully before use.

1. Adapter Preparation

1) Primers for Illumina platform (names and sequences):

Primer A: 5'-phos-CTGTCTTATACACATCT-NH2-3'

Primer B: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3'

Primer C: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3'

2) Dissolve Primer A, Primer B, and Primer C in Annealing Buffer to a concentration of 100 μM.

3) Prepare the following reaction mixtures, respectively:

4) Vortex and mix Reactions 1 and 2 thoroughly, then briefly centrifuge to collect the solution at the bottom of the tubes. Place the tubes in a PCR cycler and run the following program: Heat the lid to 105°C (On), then heat the block to 94°C for 2 min. Once the time is up, stop the program. Do not open the lid; allow the PCR tubes to cool naturally inside the instrument for 2 h, followed by incubation at 4°C for 5 min.

5) After cooling, mix Reactions 1 and 2 in equal volumes. Label this mixture as "Adapter Mix" and store it at -25°C to -15°C.

2. Transposome Formation

1) Prepare the following reaction mixture:

[Note]: *Adapter mix should be prepared by the user according to the experimental purpose and sequencing platform.

2) Reaction conditions: Mix thoroughly by gently pipetting up and down. Incubate at 25°C for 1 h (with the heated lid turned off). Label the reaction product as "Tn5 Mix". It can be used directly in library preparation or stored at -25°C to -15°C.

3. DNA Fragmentation Test

1) Prepare the following reaction mixture:

[Note]: *The larger the amount of input DNA, the longer the average length of the fragmented products; conversely, the smaller the amount of input DNA, the shorter the average length.
**To increase the degree of fragmentation, increase the amount of Tn5 Mix used; conversely, reduce the enzyme amount.
2) Fragmentation Reaction Procedure
Mix thoroughly by gentle pipetting or vortexing, then briefly centrifuge. Perform the fragmentation reaction according to the program outlined in the table below.

3) Termination of DNA Fragmentation

Prepare the following reaction mixture. Add the components listed in the table below to the fragmentation product described above, and mix thoroughly by gently pipetting up and down 20 times.

Carry out the termination reaction according to the reaction program shown in the table below.

4) After the sample temperature drops to 4°C, remove the PCR tubes and proceed with the purification of the fragmented products. Purification can be performed using 1.2× magnetic beads; Hieff NGS^TM DNA Selection Beads (Cat#12601ES) are recommended. Finally, elute the fragmented products with 21 μL of ddH₂O.

4. Quality Control

1). Measure the concentration using Qubit.

2). Analyze the size distribution using the Agilent Technologies 2100 Bioanalyzer.

3) Amplification of Fragmented Products

Amplification can be performed using reagents suitable for the specific experimental purpose and sequencing platform.

Notes

1. This product is for research use only.

2. Please operate with lab coats and disposable gloves，for your safety.