Zero Blunt® PCR cloning kits contain linearized pCR™-Blunt vector, ExpressLink™ T4 DNA ligase , 5X ExpressLink™ T4 DNA ligase buffer, control template, dNTPs, sterile water, and M13 forward and reverse primers. Competent cell kits contain One Shot® chemically competent E. coli, S.O.C. medium, and a supercoiled control plasmid.

Store the One Shot® E. coli at -80°C. Store all other components at -20°C. All reagents are guaranteed stable for 6 months when properly stored.

描述

The Zero Blunt® PCR Cloning Kit offers an easy method for high-efficiency (>80%) cloning of blunt-end PCR products amplified with proof-reading, thermostable DNA polymerases. The Zero Blunt® PCR Cloning Kit uses the multipurpose cloning vector pCR™-Blunt and ExpressLink™ T4 DNA Ligase to generate a ligation product in a five-minute, room-temperature ligation step.

Features of the Zero Blunt® Cloning® Kit with pCR™-Blunt vector:
• Fast & convenient—5-minute, room-temperature ligation
• Efficient—ccdB gene for positive selection results in >80% clones with correct insert
• Flexible—choice of kanamycin or Zeocin™ resistance for flexible antibiotic selection

The pCR™-Blunt vector provides:
• EcoR I sites flanking the PCR product insertion site for excision of inserts
• T7 promoter/primer site for in vitro RNA transcription and sequencing
• M13 forward and reverse primer sites for sequencing or PCR screening

How Zero Blunt® PCR Cloning Works
The Zero Blunt® PCR Cloning Kit is designed to clone blunt PCR fragments (or any blunt DNA fragment) with a low background of non-recombinants. The pCR™-Blunt vector contains the lethal E. coliccdB gene fused to the C-terminus of LacZα (Bernard et al., 1994). Ligation of a blunt PCR fragment disrupts expression of the lacZα-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed when the transformation mixture is plated.

Kit Configurations
The Zero Blunt® PCR Cloning Kit is offered in a variety of configurations: with One Shot® TOPO10 Chemically Competent E. coli (K2700-20 and K2700-40) and without competent cells (K2750-20 and K2750-40) in 20- and 40- reaction kit sizes.

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