NZY-A PCR cloning kit

NZY-A PCR cloning kit was designed to allow the direct cloning of PCR products with 3´-A overhangs, which result from amplifications using non-proofreading DNA polymerases, such as NZYTaq DNA polymerase.

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 the plasmid by preventing re-circularization 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:

• 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

Protocol overview:

• Specifications:
• Cloning method:    TA PCR
• Ligation reaction time:    1 hour
• Ligation reaction temperature:    room temperature
• Storage conditions:    Store competent cells at -80 °C. Other kit components may be stored at -20 °C or at -80 °C
• Shipping conditions:    Shipped at dry ice

Components:

• NZY-A Buffer (2x)
• pNZY28-A vector (50 ng/µL)
• T4 DNA Ligase (2 U/µL)
• NZY-A positive control insert (50 ng/µL)
• NZYStar Competent Cells (*)
• Competent Cells Control Plasmid (0.1 ng/µL) (*)

(*) only provided in MB05301 kit