NZY-A Speedy PCR cloning kit

24 ligations + competent cells

NZY-A Speedy PCR cloning kit was designed to carry out fast and efficient cloning of PCR products with 3´-A overhangs, which result from amplifications using non-proofreading DNA polymerases such as NZYTaq DNA polymerase. This methodology combines the efficiency of an improved ligation buffer with the speed of Speedy Ligase to allow a rapid ligation between the vector and the PCR product in only 5 minutes at room temperature (18-25 °C).

The cloning vector was prepared by cutting NZYTech´s pNZY28 with EcoR V and adding a 3´ terminal thymidine at both ends. These single 3´-T overhangs improve the efficiency of ligation of a PCR product into plasmids by preventing recircularization of the vector and providing compatible overhangs for PCR products generated by non-proofreading thermostable polymerases. Vector pNZY28 contains multiple restriction sites within the multiple cloning region. However, vector digestion with EcoR I or BamH I allows the release of the PCR product since the vector cloning region is flanked by recognition sites of both enzymes.

Features:

• Fast and easy TA cloning procedure
• High-efficiency cloning (>90% of positive clones)
• No requirement for time-consuming  restriction digests
• Possibility of blue/white screening

Applications:

• Cloning of A overhangs PCR products
• Possibility of clone blunt-ended PCR products after an A-tailing procedure

Specifications:

• Cloning method:  TA PCR
• Ligation reaction time:  5 min
• Ligation reaction temperature:  room temperature

Components:

• NZY-A Speedy Buffer (4x)
• pNZY28-A vector (50 ng/µL)
• Speedy Ligase (5 U/µL)
• NZY-A positive control insert (50 ng/µL)
• NZYStar Competent Cells (*)
• Competent Cells Control Plasmid (0.1 ng/µL) (*)

(*) only provided in MB13701 kit

Protocol