Q5® Site-Directed Mutagenesis Kit
The Q5® Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours.
- Non-overlapping primer design ensures robust, exponential amplification, generating a high percentage of desired mutations from a wide range of templates
- Intramolecular ligation and transformation into NEB high-efficiency competent cells results in high colony yield
- Extremely low error rate of Q5 Hot Start High-Fidelity DNA Polymerase reduces screening time
- Hot start polymerase enables room temperature reaction set up
- DpnI background reduction permits a wide range of starting template concentrations
- Use of standard primers eliminates additional expenses from phosphorylated or purified oligos
- Easy-to-use PCR master mix and unique multi-enzyme KLD Mix offer convenience and quality
- Rapid and direct treatment step proceeds at room temperature in 5 minutes
- Allows the use of any chemically-competent E. coli cells suitable for cloning
- Supplied with competent cells
- Use NEBaseChanger™ tool to generate primer sequences and an annealing temperature
Overview of the Q5® Site-Directed Mutagenesis Kit
Troubleshooting tips for Q5 Site-Directed Mutagenesis Kit
Quick Tips - How can I improve the efficiency of the DpnI digestion?
Quick Tips - Why do I have so many wildtype colonies?
Quick Tips - Can I make multiple mutations with the Q5 site-directed mutagenesis kit?
Quick Tips - How do I determine the best annealing temperature for my primers?
The Q5 Site-Directed Mutagenesis Kit enables rapid, site-specific mutagenesis of double-stranded plasmid DNA in less than 2 hours (Figure 1). The kit utilizes the robust Q5 Hot Start High-Fidelity DNA Polymerase along with custom mutagenic primers to create insertions, deletions and substitutions in a wide variety of plasmids. After PCR, the amplified material is added directly to a unique Kinase-Ligase-DpnI (KLD) enzyme mix for rapid (5 minutes), room temperature circularization and template removal (Figure 2). Transformation into high-efficiency NEB 5-alpha Competent E. coli, provided with the kit, ensures robust results with plasmids up to at least 20 kb in length.
The following reagents are supplied with this product:
Store at (°C) Concentration KLD Reaction Buffer -20 2 X SOC Outgrowth Medium 4 NEB® 5-alpha Competent E. coli (High Efficiency) -80 Q5® Hot Start High-Fidelity 2X Master Mix -20 2 X KLD Enzyme Mix -20 10 X Control SDM Plasmid -20 5 μg/ml pUC19 Vector -20 0.05 ng/μl Control SDM Primer Mix -20 10 μM
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