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Cat. No.: QBK-20 (for 20T)

Cat. No.: QBK-100 (for 100T)

Description

Quick Blunting Kit is a reagent kit used for the rapid conversion of double-stranded DNA into blunt-ended DNA suitable for subsequent blunt-end ligation reactions. It is also known as Quick Blunting Kit for Blunt-end Ligation or End Repair Kit for Blunt-end Ligation. This kit allows the transformation of double-stranded DNA with 5' or 3' overhangs into blunt-ended DNA with 5' phosphate and 3' hydroxyl groups, facilitating efficient ligation with blunt-end vectors or adapters.

The Blunting Enzyme Mix provided in this kit contains T4 DNA Polymerase (D7052), which possesses both 3'→5' exonuclease activity and 5'→3' polymerase activity. It can trim and fill in the ends of double-stranded DNA, resulting in blunt ends. The T4 Polynucleotide Kinase (D7098) included in the Blunting Enzyme Mix can phosphorylate the 5' ends of the blunt-ended DNA and catalyze the conversion of the 3' phosphate to a 3' hydroxyl group through its 3' phosphatase activity. This ensures compatibility for subsequent ligation reactions with vectors or adapters.

This optimized kit allows for the blunting, 5' phosphorylation, and 3' hydroxylation of 5 µg of DNA in a single reaction.

Application

This kit is used for the blunt-ending treatment of fragmented DNA resulting from enzyme digestion, mechanical shearing, etc. It enables subsequent ligation with plasmids, cosmids, fosmids, BAC vectors, as well as DNA adaptors for second-generation sequencing. It can also be used for the blunt-ending of PCR products, cDNA, etc., facilitating blunt-end cloning or high-throughput sequencing adaptor ligation.

The reaction volume for this kit is 25 μl, and the small and medium pack sizes allow for approximately 20 and 100 reactions, respectively.

Precautions

• PCR products should be appropriately purified before performing the blunt-ending and 5' phosphorylation with this kit.
• In many cases, DNA generated from restriction enzyme digestion can be used directly for blunt-ending, 5' phosphorylation, and 3' dephosphorylation reactions without purification. Although there may be some differences in reaction efficiency, it will not affect blunt-end cloning. If used for high-throughput sequencing library construction, the need for purification before subsequent reactions should be considered on a case-by-case basis.
• This product is intended for scientific research use by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in residential areas.
• For your safety and health, please wear appropriate laboratory attire and disposable gloves when handling.

Storage

The minimum shelf life is 1 year at -20°C.