Fast MultiSite Mutagenesis System is used for generating mutated PCR fragments by introducing mutation sites on overlapping regions. High fidelity TransStart® FastPfu PCR SuperMix is included for amplification. This kit uses proprietary assembly mix and homologous recombination to seamlessly assemble up to six mutagenesis fragments.
• Fast: Amplified with fast & high-fidelity 2×TransStart® FastPfu PCR SuperMix; only 15 minutes for recombination.
• Flexible: Able to be cloned into any site to realize single-site/ multi-site, continuous/non-continuous mutagenesis.
• Efficient: >90% mutagenesis efficiency.
• Both primers contain overlapping region at the 5’ ends and extension region at the 3’ ends, with mutation site on overlapping region, as shown in figure 1.
• Primer length: Both primers (forward and reverse) should be approximately at 25-40 nucleotides in length, excluding the mutation site. Primers should have an overlapping region of 15-25 nucleotides and have an extension region of at least 10 nucleotides.
Amplified PCR products can be checked by electrophoresis with 10 μl of the product on a 1% agarose gel.
• Digestion of PCR Product with DMT
• Purification of PCR products
•Add 2 μl of assembly products into 50 μl of DMT Chemically Competent Cell (DNA should be added immediately after thawing the cells on ice) and mix by tapping gently. Incubate on ice for 20-30 minutes.
•Heat-shock at 42oC for exactly 45 seconds, quickly remove from 42oC water bath and place on ice for 2 minutes.
•Add 250 μl of SOC or LB medium (pre-warm to room temperature), and incubate at 37oC for 1 hour with shaking at 200 rpm.
•Pre-warm a selective plate at 37oC for 30 minutes.
•Spread 100-200 μl of transformants on the plate and incubate at 37oC overnight
Positive Clone Analysis
Analyze the clones by sequencing.
We suggest performing 25 cycles for PCR. For low yield PCR products, we suggest using up to 30 cycles.
FM201-01 (10 rxns)
2×TransStart® FastPfu PCR SuperMix
DMT Enzyme (10 units/μl)
DMT Chemically Competent Cell
|Citations & references|
|Activation of GSNOR transcription by NF-κB negatively regulates NGF-induced PC12 differentiation||Informa healthcare||Kaiyuan Wu, et al.||2014 Mar||http://informahealthcare.com/doi/abs/10.3109/10715762.2014.906743|
|A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase||The FASEB Journal||5.704||Xiaoyan Liu, et al.||2014 Apr||http://www.fasebj.org/content/early/2014/04/23/fj.13-246850.abstract|
|Tyrosine 402 Phosphorylation of Pyk2 Is Involved in Ionomycin-Induced Neurotransmitter Release||PLoS ONE||3.73||Zhao Zhang, et al.||2014 Apr||http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0094574|
|Lipid raft-associated β-adducin is required for PSGL-1-mediated neutrophil rolling on P-selectin||Journal of Leukocyte Biolog||4.289||Tingshuang Xu, et al.||2015 Feb||http://www.jleukbio.org/content/97/2/297.short|
|Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device||Applied Microbiology and Biotechnology||3.337||Junming Wang, et al.||2015 May||http://link.springer.com/article/10.1007/s00253-015-6615-0|
|Crystal structure of the polo-box domain of polo-like kinase 2||Biochemical and Biophysical Research Communications||2.297||Hong-Mei Shan, et al.||2014 Dec||http://www.sciencedirect.com/science/article/pii/S0006291X14022098|
|Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling||Cellular Signalling||4.304||Peng Yang, et al.||2014 Sep||http://www.sciencedirect.com/science/article/pii/S0898656814001223|