USB Change-IT Multiple Mutation Site Directed Mutagenesis Kit

Designed to create single or multiple oligonucleotide-directed sequence changes in plasmids.

• Based on highly efficient exponential amplification using non-overlapping primers; eliminates primer-dimer formation
• Create single or multiple mutations in plasmids
• Create mutations in plasmids as large as 15 Kb
• Create insertions, base changes, or deletions
• Create deletions as large as 300 bp
• Encode multiple base changes in a single primer
• Three step process: amplification, DpnI digestion, and transformation
• Single day method

Mutations are created using a variation of ligation during-amplification in which the plasmid is PCR-amplified with phosphorylated primers and the product ends are ligated together to convert the annealed DNA strands into closed circular DNA molecules. These closed circular DNA molecules then become template molecules in subsequent rounds of the PCR amplification. The desired mutations (insertions, deletions, or substitutions) are created by incorporating them into the primer sequence. USB’s FideliTaq Polymerase is used to ensure faithful duplication of the plasmid sequence and successful longdistance PCR. Thermostable DNA ligase is utilized for efficient creation of circular plasmid DNA during each round of PCR amplification. Together these enzymes generate exponentially amplified, circular, mutated plasmid DNA. To enhance selection of the mutated plasmid from the parental plasmid, the PCR amplification reaction is digested with Dpn I prior to transformation into competent cells.

Product Code: USB-78480-20reactions 

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