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Your present location:HomeProductsCloning and Mutagenesis SystemCloning Vectors / pEASY®-T3 Cloning Kit
  • Product Name: pEASY®-T3 Cloning Kit
  • Catalog Number: CT301
  • Storage: Competent Cell at -70°C for six months; others at -20°C for one year
  • 币种:  
    • Specification: 20 rxns 60 rxns
  • Quantity:  

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Overview

Description

pEASY ®- T3 Cloning Vector provides dual EcoR I and dual Not I enzyme sites. It is designed for cloning and sequencing Taq-amplified PCR products. The cloned insert can be released from a single enzyme digestion.

•5 minutes fast ligation of Taq-amplified PCR products.

•Ampicillin resistance genes for selection.

•Easy blue/white selection.

•T7 promoter, SP6 promoter, M13 forward and M13 reverse primers for sequencing.

•T7 promoter and SP6 promoter for in vitro transcription.

•Trans1-T1 Phage Pesistant Chemically Competent Cells, high transformation efficiency (>109 cfu/μg pUC19 DNA) and fast growing.

 

Preparation

1.Primer requirement: primer cannot be phosphorylated

2.PCR Enzyme: Taq DNA polymerases

3.Reaction conditions: in order to ensure the integrity of amplification products, 5-10 minutes of post-extension step is required. After amplification reaction, use agarose gel electrophoresis to verify the quality and quantity of PCR product

 

Reaction System

Add following components into a microcentrifuge tube.

PCR products 0.5-4 μl (can be increased or reduced based on PCR product yield, no more than 4 μl)

pEASY ®- T3 Cloning Vector 1 μl

Gently mix well, incubate at room temperature (20oC-37oC) for 5 minutes. After reaction, place the tube on ice.

1. Optimal amount of insert

Molar ratio of vector to insert = 1:7 (1 kb, ~20 ng; 2 kb, ~40 ng)

2.Optimal volume of vector: 1 μl(10 ng)

3.Optimal reaction volume: 3~5 μl

4.Optimal incubation time

(1)0.1~1 kb (including 1 kb): 5~10 minutes

(2)1~2 kb (including 2 kb): 10~15 minutes

(3)2~3 kb (including 3 kb): 15~20 minutes

(4)≥3 kb: 20~30 minutes

Use the maximum incubation time if the insert is gel purified.

5.Optimal incubation temperature: for most PCR inserts, the optimal temperature is about 25oC; for some PCR inserts, optimal results can be achieved with higher temperature (up to 37oC).

 

Transformation

1.Add the ligated products to 50 μl of Trans1-T1 Phage Resistant Chemically Competent Cell and mix gently (do not mix by pipetting up and down).

2.Incubate on ice for 20~30 minutes.

3.Heat-shock the cells at 42oC for 30 seconds.

4.Immediately place the tube on ice for 2 minutes.

5.Add 250 μl of room temperature SOC or LB medium. Shake the tube at 37oC (200 rpm) for 1 hour.

6.In the meantime, mix 8 μl of 500 mM IPTG with 40 μl of 20 mg/ml X-gal. Spread them evenly onto a selective LB plate. Place the plate at 37oC for 30 minutes.

7.Spread 200 μl or all transformants on the pre-warmed plate. Incubate at 37oC overnight.

 

Contents& storage

 

Component

CT301-01

CT301-02

(20 rxns)

(60 rxns)

 

pEASY ®-T3 Cloning Vector (10 ng/μl)

20 µl

3×20 µl

Control Template (5 ng/μl)

5 µl

5 µl

Control Primers (10 μM)

5 µl

5 µl

M13 Forward Primer (10 μM)

50 µl

150 µl

M13 Reverse Primer (10 μM)

50 µl

150 µl

Trans1-T1 Phage Resistant

Chemically Competent Cell

10×100 μl

30×100 μl

 

Citations & references
Literature Journal IF Author Date Link
Evolution and Expression Plasticity of Opsin Genes in a Fig Pollinator, Ceratosolen solmsi PLoS One 3.73 Wang B. et al. 2013 http://www.ncbi.nlm.nih.gov/pubmed/23342036
The impact of dissolved organic carbon on the spatial variability of methanogenic archaea communities in natural wetland ecosystems across China Applied Microbiology and Biotechnology 3.689 Liu D, et al. 2012 Oct http://www.ncbi.nlm.nih.gov/pubmed/22218772
Arsenic induces functional re-expression of estrogen receptor α by demethylation of DNA in estrogen receptor-negative human breast cancer PLoS One 3.73 Du J, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22558281
Complex evolution in Arundinarieae (Poaceae: Bambusoideae): incongruence between plastid and nuclear GBSSI gene phylogenies Molecular Phylogenetics and Evolution 4.066 Zhang YX, et al. 2012 Jun http://www.ncbi.nlm.nih.gov/pubmed/22415014
DsbM, a novel disulfide oxidoreductase affects aminoglycoside resistance in Pseudomonas aeruginosa by OxyR-regulated response Journal of Microbiology 1.276 Wang X, et al. 2012 Dec http://link.springer.com/article/10.1007/s12275-012-2177-3
Sinomyces: a new genus of anamorphic Pleosporaceae Fungal Biology 2.082 Wang Y, et al. 2011 Feb http://www.ncbi.nlm.nih.gov/pubmed/21315316
Identification of ethylene-responsive genes in ethrel-treated shoot apices of cucumber by suppression subtractive hybridization Plant Molecular Biology Reporter 5.319 Jin X, et al. 2011 Dec

http://link.springer.com/article/10.1007/s11105-011-0304-7

Three novel species of Stemphylium from Sinkiang, China: their morphological and molecular characterization Mycological Progress 1.606 Pei YF, et al. 2011 May http://link.springer.com/article/10.1007/s11557-010-0686-1
Molecular and morphological description of two new species of Stemphylium from China and France Mycologia 2.11 Wang Y, et al. 2010 May-Jun http://www.ncbi.nlm.nih.gov/pubmed/20524602
Ulocladium cantlous sp. nov. isolated from northwestern China: its morphology and molecular phylogenetic position Mycologia 2.11 Wang Y, et al. 2010 Mar-Apr http://www.ncbi.nlm.nih.gov/pubmed/20361504
Molecular characterization, immunohistochemical localization and expression of a ribosomal protein L17 gene from Apis cerana cerana Insect Biochemistry and Physiology 1.515 Fei Meng, et al. 2010 Oct http://onlinelibrary.wiley.com/doi/10.1002/arch.20386/abstract
The complete mitochondrial genome of Spilonota lechriaspis Meyrick (Lepidoptera: Tortricidae) Molecular Biology Reports 2.506 Jin-Liang Zhao, et al. 2011 Aug http://link.springer.com/article/10.1007/s11033-010-0491-6
Halomonas songnenensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils IJSEM   Juquan Jiang,et al. 2014 May http://ijs.sgmjournals.org/content/64/Pt_5/1662.short
Characterization of a mitochondrial manganese superoxide dismutase gene from Apis cerana cerana and its role in oxidative stress Journal of Insect Physiology 2.379 Haihong Jia, et al. 2014 Jan http://www.sciencedirect.com/science/article/pii/S002219101300231X
Cryptic diversity in host-associated populations of Tetra pinnatifidae (Acari: Eriophyoidea): What do morphometric, mitochondrial and nuclear data reveal and conceal? Bulletin of Entomological Research 1.987 Hao-Sen Li, et al. 2014 Apr

http://journals.cambridge.org/action/displayAbstract?fromPage=

online&aid=9192274&fileId=S0007485313000746

Identification of Rhizoctonia solani AG1-IB as a causal agent of leaf blight in Cynanchum paniculatum in China Journal of General Plant Pathology 0.893 Ai Xin Wang, et al. 2014 Jan http://link.springer.com/article/10.1007/s10327-013-0489-8
MOLECULAR CHARACTERIZATION OF SOLUBLE AND MEMBRANE-BOUND TREHALASES IN THE COTTON MIRID BUG, Apolygus lucorum Archives of Insect Biochemistry and Physiology 1.515 Yongan Tan, et al. 2014 Jun

http://onlinelibrary.wiley.com/doi/10.1002/arch.21166/abstract?

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RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furcifera Pesticide Biochemistry and Physiology 2.111 Yao Yang, et al. 2014 Jan http://www.sciencedirect.com/science/article/pii/S0048357513001922
Pollen and seed transmission of Cucumber green mottle mosaic virus in cucumber Plant Pathology 2.729 H.W.Liu, et al. 2014 Feb

http://onlinelibrary.wiley.com/doi/10.1111/ppa.12065/full

Biochemical functions of the glutathione transferase supergene family of Larix kaempferi Plant Physiology and Biochemistry 2.775 Qi Yang, et al. 2014 Apr http://www.sciencedirect.com/science/article/pii/S0981942814000473
RNA interference-mediated silencing of a Halloween gene spookier affects nymph performance in the small brown planthopper Laodelphax striatellus Insect Science 1.786 Shuang Jia, et al. 2014 Apr

http://onlinelibrary.wiley.com/doi/10.1111/1744-7917.12087/abstract;jsessionid=CCB2CD4081BEAA2C0

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Differential Expression Patterns in Chemosensory and Non-Chemosensory Tissues of Putative Chemosensory
Genes Identified by Transcriptome Analysis of Insect Pest the Purple Stem Borer Sesamia inferens (Walker)
PLOS one 3.7 Zhang, YN,et al. 2013 Aug http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069715
A novel bifunctional pectinase from Penicilliumoxalicum SX6with separate pectin methylesterase and polygalacturonasecatalytic domains Appl Microbiol Biotechnol 3.689 Tao Tu, et al. 2014 Jan http://link.springer.com/article/10.1007/s00253-014-5533-x
Two Origins of Blastemal Progenitors Define Blastemal
Regeneration of Zebrafish Lower Jaw
PloSOne 3.54 Xuelong Wang, et al. 2014 Dec http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0045380
Two Metallothionein Genes in Oxya chinensis: Molecular
Characteristics, Expression Patterns and Roles in Heavy
Metal Stress
PloSOne 3.54 Yaoming Liu, et al. 2014 Nov http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0112759
High Level Expression of a Novel Family 3 Neutral β-Xylosidase from Humicola insolens Y1 with High Tolerance to D-Xylose PloSOne 3.54 Wei Xia, et al. 2015 Feb http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0117578
Electrical signals generated by soil microorganisms in microbial fuel cells respond linearly to soil Cd 2+ pollution Geoderma 2.772 Yun-Bin Jiang, et al. 2015 Apr Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control, School of Geography Science, Nanjing Normal University, 210023 Nanjing, China
Small C-terminal Domain Phosphatase 3 Dephosphorylates the Linker Sites of R-Smads to Ensure TGFβ-mediated Germ Layer Induction in Xenopus Embryos The Journal of Biological Chemistry 4.573 Guanni Sun, et al. 2015 May http://www.jbc.org/content/early/2015/05/26/jbc.M115.655605.short
Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity Journal of Bioscience and Bioengineering 1.884 Caihong Wang, et al. 2015 Jun http://www.sciencedirect.com/science/article/pii/S1389172315001978
Post-translational Modifications of Baculovirus Protamine-like Protein P6.9 and the Significance of Its Hyperphosphorylation for Viral Very Late Gene Hyperexpression Journal of Virology 4.439 Ao Li, et al. 2015 May http://jvi.asm.org/content/early/2015/05/11/JVI.00333-15.abstract
High-yield production of a low-temperature-active polygalacturonase for papaya juice clarification Food chemistry 3.334 Tao Tu,et al. 2013 Jun http://www.sciencedirect.com/science/article/pii/S0308814613007383
Genome-wide screening and functional analysis identify a large number of long noncoding RNAs involved in the sexual reproduction of rice Genome Biology 10.465 Yu-Chan Zhang, et al. 2014 Dec http://www.biomedcentral.com/content/pdf/s13059-014-0512-1.pdf
Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching. Nature Biotechnology 39.08 Yu-Chan Zhang, et al. 2013 Sep http://www.nature.com/nbt/journal/v31/n9/full/nbt.2646.html
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