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  • Product Name: Fast MultiSite Mutagenesis System
  • Catalog Number: FM201
  • Date: 2016-01-04
  • Views: 13
  • Sold amount:0Piece
    • Specification: 10 rxns
  • Quantity:  

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Overview

Description

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.

 

Primer Design

• 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.

 

Electrophoresis Analysis

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

• Purification of PCR products
For PCR product with a single expected band, it is suggested to use PCR Purification Kit to purify PCR products; for PCR product with multibands, we suggest using Quick Gel Extraction Kit to purify PCR products.

 

Transformation

•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.

 

Notes

We suggest performing 25 cycles for PCR. For low yield PCR products, we suggest using up to 30 cycles.

 

Contents& storage

 

Kit Contents
Component

FM201-01 (10 rxns)

2×TransStart® FastPfu PCR SuperMix

1 ml

DMT Enzyme (10 units/μl)

30 μl

2×Assembly Mix

50 μl

DMT Chemically Competent Cell

10×50 μl

ddH2O

1 ml

 

Citations & references
Literature Journal IF Author Date Link
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

 

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