Thermo Scientific™ Phusion™ Site-Directed Mutagenesis Kit with DH10B Competent Cells

Catalog No.	Quantity
F542	20 Reactions

Catalog No. F542 Supplier Thermo Scientific™ Supplier No. F542

Description

A versatile and efficient tool for introducing point mutations, insertions, or deletions into any type of plasmid DNA. With this kit the entire plasmid is amplified using phosphorylated primers that introduce the desired changes.

• This package includes a Phusion Site-Directed Mutagenesis Kit and DH10B Competent Cells that are optimized for use with the kit
• Phusion Site-Directed Mutagenesis Kit:
  • With this kit the entire plasmid is amplified using phosphorylated primers that introduce the desired changes
  • The amplified, linear PCR product, containing the desired mutation, is circularized in a 5-minute ligation reaction with T4 DNA Ligase. The resulting plasmid can be then transformed into the E.coli cells. DH10B Competent Cells are recommended for use with the kit.
  • The optimal annealing temperature for Phusion DNA polymerases may differ significantly from that of Taq-based polymerases. For optimal results start by accurately calculating your Tm with our Tm calculator.
  • Robust and reliable exponential amplification method
  • No requirements, such as special vectors, restriction sites, or methylation status for the target plasmid
  • No need to destroy the starting template in a separate step
  • Phusion Hot Start II High Fidelity DNA Polymerase minimizes unwanted secondary mutations\
  • Amplification of large plasmids up to 10 kb
  • Hot start modification of the polymerase prevents amplification of non-specific products and unwanted degradation of primers prior to first cycle of PCR
  • T4 DNA Ligase included in the kit; no purification steps before or after ligation
• DH10B Competent Cells
  • High efficiency, chemically competent E.coli cells
  • The DH10B strain is suitable for cloning DNA containing methylcytosine and methyladenine, allowing both prokaryotic and eukaryotic genomic DNA to be cloned efficiently
  • These cells are ideal for the construction of cDNA libraries using plasmid-derived vectors
  • Genotype: F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ (ara-leu)7697 galU galK λ– rpsL(StrR) nupG

Specifications

• Cell Type: DH10B
• Content And Storage: Box 1
  • 10 x DH10B Competent Cells, 100 μL
  • pUC19 DNA, 50 μL, 100 pg/μL
  • S.O.C. Medium, 5 mL
  Store at -80°C.
  
  Box 2
  • Phusion Hot Start II DNA Polymerase, 10 μL, 2 U/μL
  • Phusion HF Buffer (5X), 1.5 mL
  • dNTP Mix, 10 mM each, 20 μL
  • FastDigest DpnI, 20 μL
  • Control plasmid (in TE buffer), 5 ng/μL
  • Control primer mix (25 μM each of 5' phosphorylated primers), 20 μL
  • T4 DNA Ligase, 15 μL
  • Rapid Ligation Buffer (5X), 200 μL
  Store at -20°C.
• Format: Kit
• Sample Type: PCR Products
• Efficiency: ≥1 x 10^9
• Enzyme: Maxima H Minus
• For Use With (Application): Mutagenesis
• Mutagenesis Capability: Single-Site Mutagenesis
• Product Line: Phusion, Thermo Scientific
• Product Type: Site-Directed Mutagenesis Kit
• Quantity: 20 Reactions
• Shipping Condition: Dry Ice

Frequently Asked Questions (FAQs)

Do Phusion DNA Polymerases add the non-template dependent 3'-A overhang?

Phusion DNA Polymerases generate blunt end products; therefore, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product with e.g. Taq DNA Polymerase (Cat. No. EP0401). However, before adding the overhangs it is very important to remove all the Phusion DNA Polymerase by purifying the PCR product carefully, as the proofreading activity in Phusion DNA Polymerase is very strong. Any remaining Phusion DNA Polymerase will degrade the A overhangs, thus creating blunt ends again.

Can Phusion DNA Polymerases extend at 1 second/kb?

Yes it is possible, especially when amplifying smaller amplicons. Processivity of Phusion DNA Polymerases is 10 times that of Pfu. We recommend extension times of 15 s/kb for Phusion Flash PCR Master Mix. 15 s/kb is a conservative value that we can promise to work with almost any amplicon. In many cases, significantly shorter extension times (0-5 s/kb) can be used without compromising the yield. What separates Phusion Flash DNA Polymerase from other fast polymerases is that all steps in the PCR protocol can be shortened, including annealing and denaturation. This results in extremely fast protocols as compared with any other polymerase.

How can I obtain the highest possible transformation efficiency with Thermo Scientific DH5α and DH10B competent cells?

To achieve the highest possible transformation efficiency with Thermo Scientific DH5α (Cat. No. EC0112) and DH10B (Cat. No. EC0113) competent cells, please follow the transformation protocol provided with the product and pay attention to the following details:

- Make sure to use wet well-pressed ice to maximize cold surface volume in all the transformation steps.
- Perform the steps quickly and accurately.
- Use chilled microcentrifuge tubes and bury the whole tube up to the cap in the wet ice.
- Do not exceed the recommended heat shock time and transfer the tube with cells back to the wet ice immediately.