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  • Product Name: Fast Mutagenesis System
  • Catalog Number: FM111
  • Storage: Competent Cell at -70°C for six months; others at -20°C for two years
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Overview

Description

• Primers anneal to the DNA template, mutant strands are synthesized with 2×TransStart® FastPfu PCR SuperMix.

• In vitro digestion of non-mutated parental plasmid (methylated plasmid) with DMT enzyme and in vivo degradation of non-mutated parental plasmid (methylated plasmid) with DMT Chemically Competent Cell, so as to efficiently select mutant clones.

 

Highlights

• Mutation sites on both primers to improve mutation efficiency.

• Partially overlapping primers for exponential DNA amplification.

• Fast (4 kb/min) and high fidelity ( 54-fold fidelity as compared to EasyTaq® DNA Polymerase) 2xTransStart® FastPfu PCR SuperMix for DNA amplification.

• Double digestions (in vitro and in vivo) of parental plasmids to enhance mutation efficiency.

 

Primer Design

• Both primers (forward and reverse) should be approximately at 25-30 nucleotides in length.

• Primers should have an overlapping region of 15-20 nucleotides for exponential amplification.

• Primers should have an extension region of at least 10 nucleotides.

• The mutation site should be located on both primers.

 

Electrophoresis Analysis

Amplification may be checked by electrophoresis with 10 μl of the product on a 1% agarose gel.

Note:Proceed with DMT enzyme digestion and transformation if the expected size product can be visualized on the gel.

 

Digestion of PCR Product

Add 1 μl of DMT enzyme into PCR product, mix thoroughly and incubate at 37oC for 1 hour.

 

Transformation

•Add 2-5 μl of DMT enzyme-treated PCR product into 50 μl of DMT Chemically Competent Cell (PCR product should be added immediately after thawing the cells on ice) and mix by tapping gently. Incubate on ice for 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/LB medium (equilibrated to room temperature), and shake at 225 rpm at 37oC for 1 hour.

•Spread 200 μl of transformants on the plate and incubate overnight (to obtain more colonies, centrifuge the transformation vial at 4000 rpm for 1 minute, discard a portion of supernatant and keep 100-150 μl of it. Gently tapping to suspend the cells, plate all the cells and incubate overnight).

 

 

Notes

•If no colony or low numbers of colonies are observed, it is suggested to purify the DMT enzyme-treated DNA with PCR purification kit, then perform transformation with 2-5 μl of the purified product.

•If use the control plasmid (4.5 kb) to test the mutation efficiency, spread cells on agar plates containing 8 μl of 500 mM IPTG and 40 μl of 40 mg/ml X-gal, successful transformation is indicated by the observation of blue colonies.

 

Contents& storage

 

Component

FM111-01 (10 rxns)

FM111-02 (20 rxns)

2×TransStart® FastPfu PCR SuperMix

250 µl

500µl

DMT Enzyme (10 units/μl)

10 µl

20µl

DMT Chemically Competent Cell

10×50 μl

20×50 μl

ddH2O

1 ml

1 ml

SControl Plasmid (5 ng/µl)

10 µl

20µl

SControl Primers (10 µM )

10 µl

20µl

 

Citations & references
Literature Journal IF Author Date Link
Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification Molecular and Cellular Proteomics 7.251 Wang C, et al. 2013 Sep http://www.ncbi.nlm.nih.gov/pubmed/24019147
Mutation in AQP5, Encoding Aquaporin 5, Causes Palmoplantar Keratoderma Bothnia Type The Journal of Investigative Dermatology 6.193 Cao X, et al. 2013 Jul http://www.ncbi.nlm.nih.gov/pubmed/23867895
miR-200b suppresses invasiveness and modulates the cytoskeletal and adhesive machinery in esophageal squamous cell carcinoma cells via targeting Kindlin-2 Carcinogenesis 5.635 Zhang HF, et al. 2013 Sep http://www.ncbi.nlm.nih.gov/pubmed/24064224
Gestational Hypoxia Induces Sex-Differential Methylation of Crhr1 Linked to Anxiety-like Behavior Molecular Neurobiology 5.471 Wang X, et al. 2013 Mar http://www.ncbi.nlm.nih.gov/pubmed/23529784
Chronic Inhibition of Cyclic Guanosine Monophosphate-Specific Phosphodiesterase 5 Suppresses Endoplasmic Reticulum Stress in Heart Failure British Journal of Pharmacology 5.067 Gong W, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/24032459
Neuroglobin, a Novel Intracellular Hexa-Coordinated Globin, Functions as a Tumor Suppressor in Hepatocellular Carcinoma via Raf/MAPK/Erk Molecular Pharmacology 4.411 Zhang J, et al. 2013 May http://www.ncbi.nlm.nih.gov/pubmed/23478801
The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes Cellular Signalling 4.304 Zhang H, et al. 2013 Jul http://www.ncbi.nlm.nih.gov/pubmed/23567262
Distinct post-transcriptional regulation of Igfbp1 gene by hypoxia in lowland mouse and Qinghai-Tibet plateau root vole Microtus oeconomus Molecular and Cellular Endocrinology 4.039 Zhang S, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/23748030
Stability and iron oxidation properties of a novel homopolymeric plant ferritin from adzuki bean seeds: A comparative analysis with recombinant soybean seed H-1 chain ferritin Biochimica et Biophysica Acta 3.848 Li M, et al. 2013 Apr http://www.ncbi.nlm.nih.gov/pubmed/23313843
Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalyzing the synthesis of ethylene in Agaricus bisporus Biochimica et Biophysica Acta 3.848 Meng D, et al. 2013 Sep http://www.ncbi.nlm.nih.gov/pubmed/24016603
FoxM1 Promotes Glioma Cells Progression by Up-Regulating Anxa1 Expression PLoS One 3.73 Cheng SX, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/23991102
Functional consequences of a novel point mutation in the CYP21A2 gene identified in a Chinese Han patient with nonclassic 21-hydroxylase deficiency Clinical Endocrinology 3.396 Chu X, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/23927611
GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress Plant Molecular Biology 3.518 Ni Z, et al. 2013 May http://www.ncbi.nlm.nih.gov/pubmed/23483290
FoxQ1 Promotes Glioma Cells Proliferation and Migration by Regulating NRXN3 Expression PLoS One 3.73 Sun HT, et al. 2013 http://www.ncbi.nlm.nih.gov/pubmed/23383267
AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth Molecular Plant 6.126 Chang F, et al. 2013 Jul http://www.ncbi.nlm.nih.gov/pubmed/23024212
Novel POMGnT1 mutations cause muscle-eye-brain disease in Chinese patients Molecular Genetics and Genomics 2.881 Jiao H, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/23689641
WNT5A/JNK Signaling Regulates Pancreatic Cancer Cells Migration by Phosphorylating Paxillin Pancreatology 2.043 Wei W, et al. 2013 Jul-Aug http://www.ncbi.nlm.nih.gov/pubmed/23890137
Exome sequencing reveals mutations in TRPV3 as a cause of Olmsted syndrome American Journal of Human Genetics 11.202 Lin Z, et al. 2012 Mar http://www.ncbi.nlm.nih.gov/pubmed/22405088
Enhancing production of bio-isoprene using hybrid MVA pathway and isoprene synthase in E. coli PLoS One 3.73 Yang J, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22558074
Let-7b inhibits human cancer phenotype by targeting cytochrome P450 epoxygenase 2J2 PLoS One 3.73 Chen F, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22761738
Direct observation of the uptake of outer membrane proteins by the periplasmic chaperone Skp PLoS One 3.73 Lyu ZX, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/23049938
Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus PloS Biology 12.69 Xu S, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22448144
Hypoxia-induced up-regulation of aquaporin-1 protein in prostate cancer cells in a p38-dependent manner Cellular Physiology and Biochemistry 3.415 Tie L, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22415096
Identification and characterization of a highly conserved crenarchaeal protein lysine methyltransferase with broad substrate specificity Journal of Bacteriology 3.194 Chu Y, et al. 2012 Dec http://www.ncbi.nlm.nih.gov/pubmed/23086207
Structural and catalytic role of two conserved tyrosines in Delta-class glutathione S-transferase from Locusta migratoria Archives of Insect Biochemistry and Physiology 1.515 Zhang X, et al. 2012 Jul http://www.ncbi.nlm.nih.gov/pubmed/22581614
Functional analysis and molecular docking identify two active short-chain prenyltransferases in the green peach aphid, Myzus persicae Archives of Insect Biochemistry and Physiology 1.515 Zhang YL, Li ZX. 2012 Oct http://www.ncbi.nlm.nih.gov/pubmed/22696503
In silico and in vitro analyses identified three amino acid residues critical to the catalysis of two aphid farnesyl diphosphate synthase The Protein Journal 1.126 Sun XF, Li ZX. 2012 Jun http://www.ncbi.nlm.nih.gov/pubmed/22588726
Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.) Plant Cell Reports 2.509 Ye R, et al. 2012 Jun http://www.ncbi.nlm.nih.gov/pubmed/22388917
PDlim2 selectively interacts with the PDZ binding motif of highly pathogenic avian H5N1 influenza A virus NS1 PLoS One 3.73 Yu J, et al. 2011 http://www.ncbi.nlm.nih.gov/pubmed/21625420
A conserved cysteine motif is critical for rice ceramide kinase activity and function PLoS One 3.73 Bi FC, et al. 2011 Mar http://www.ncbi.nlm.nih.gov/pubmed/21483860

 

LTD is a protein required for sorting light-harvesting chlorophyll-binding proteins to the chloroplast SRP pathway Nature Communications 10.015 Ouyang M, 2011 http://www.ncbi.nlm.nih.gov/pubmed/21505433
Prevalence of common mutations in the CYP17A1 gene in Chinese Han population Clinica Chimica Acta 2.85 Bao X, et al. 2011 Jun http://www.ncbi.nlm.nih.gov/pubmed/21420394
A novel low-temperature-active β-glucosidase from symbiotic Serratia sp. TN49 reveals four essential positions for substrate accommodation Applied Microbiology and Biotechnology 3.689 Zhou J, et al. 2011 Oct http://www.ncbi.nlm.nih.gov/pubmed/21559826
A new α-galactosidase from symbiotic Flavobacterium sp. TN17 reveals four residues essential for α-galactosidase activity of gastrointestinal bacteria Applied Microbiology and Biotechnology 3.689 Zhou J, et al. 2010 Dec http://www.ncbi.nlm.nih.gov/pubmed/20714719
A nonsense mutation in the Xeroderma pigmentosum complementation group F (XPF) gene is associated with gastric carcinogenesis Gene 2.196 Zhong-Hua Wei, et al. 2014 Mar http://www.sciencedirect.com/science/article/pii/S0378111914000031
miR-200b suppresses invasiveness and modulates the cytoskeletal and adhesive machinery in esophageal squamous cell carcinoma cells via targeting Kindlin-2 Carcinogenesis Integrative Cancer Research   Hai-Feng Zhang, et al. 2013 Sep http://carcin.oxfordjournals.org/content/35/2/292.short
Prediction of the key binding site of odorant-binding protein of Holotrichia oblita Faldermann (Coleoptera: Scarabaeida) Insect Molecular Biology 3.044 X.Zhuang, et al. 2014 Jun

http://onlinelibrary.wiley.com/doi/10.1111/imb.12088/abstract;jsessionid=

4FCC00A557C4D0E1DC5BCD66D98BC949.f02t03?

deniedAccessCustomisedMessage=&userIsAuthenticated=false

Functional consequences of a novel point mutation in the CYP21A2 gene identified in a Chinese Han patient with nonclassic 21-hydroxylase deficiency Clinical Endocrinology 3.396 Xiaojing Chu, et al. 2014 Jun http://onlinelibrary.wiley.com/doi/10.1111/cen.12309/abstract
Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene Molecular Biology Reports 2.506 Wei Sun, et al. 2014 Feb http://link.springer.com/article/10.1007/s11033-013-2939-y
Structural analysis of Mycobacterium tuberculosis ATP-binding cassette transporter subunit UgpB reveals specificity for glycerophosphocholine The FEBS Journal 4.25 Dunquan Jiang, et al. 2014 Jan http://onlinelibrary.wiley.com/doi/10.1111/febs.12600/full
Glutathione peroxidase 7 utilizes hydrogen peroxide generated by Ero1α to promote oxidative protein folding. Antioxidants & Redox Signaling 7.2 Wang, Lei,et al. 2014 Feb http://online.liebertpub.com/doi/abs/10.1089/ars.2013.5236

 

Whole-genome sequencing of the snub-nosed monkey provides insights into folivory and evolutionary history. Nature genetics 29.648 Xuming Zhou, et al. 2014 Dec http://www.nature.com/ng/journal/v46/n12/abs/ng.3137.html
GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress Plant Molecular Biology 4.15 Zhiyong Ni, et al. 2013 Mar http://link.springer.com/article/10.1007/s11103-013-0040-5
Identification of a novel selective agonist of PPARc with no promotion of adipogenesis and less inhibition of osteoblastogenesis Scientific Reports 5.078 Chang Liu, et al. 2015 Apr http://www.nature.com/srep/2015/150329/srep09530/full/srep09530.html
Endostatin has ATPase activity, which mediated its antiangiogenic and antitumor activities Molecular cancer therapeutics 6.1 Shan Wang, et al. 2015 Mar

http://mct.aacrjournals.org/content/14/5/1192

Pyruvate kinase M2 facilitates colon cancer cell migration via the modulation of STAT3 signalling Cellular Signalling 4.471 Yang Peng et al. 2014 May http://link.springer.com/article/10.1007/s11426-014-5095-x
Comparison of droplet digital PCR and conventional quantitative PCR for measuring EGFR gene mutation Experimental and Therapeutic Medicine 1.269 Bo Zhang, et al. 2015 Jan http://www.spandidos-publications.com/etm/9/4/1383?text=fulltext
Crystal structure of the death effector domains of caspase-8 Biochemical and Biophysical Research Communications 2.297 Chen Shen, et al. 2015 May http://www.sciencedirect.com/science/article/pii/S0006291X15009705
Structural insights into the substrate specificity of two esterases from the thermophilic Rhizomucor miehei The Journal of Lipid Research 4.421 Shaoqing Yang, et al. 2015 Jun http://www.jlr.org/content/early/2015/06/23/jlr.M060673.abstract
Identification of a novel partial agonist of liver X receptor α (LXRα) via screening Biochemical Pharmacology 4.65 Ni Li, et al. 2014 Dec http://www.ncbi.nlm.nih.gov/pubmed/25450668
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