CT301

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
LiteratureJournalIFAuthorDateLink
Evolution and Expression Plasticity of Opsin Genes in a Fig Pollinator, Ceratosolen solmsiPLoS One3.73Wang B. et al.2013http://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 ChinaApplied Microbiology and Biotechnology3.689Liu D, et al.2012 Octhttp://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 cancerPLoS One3.73Du J, et al.2012http://www.ncbi.nlm.nih.gov/pubmed/22558281
Complex evolution in Arundinarieae (Poaceae: Bambusoideae): incongruence between plastid and nuclear GBSSI gene phylogeniesMolecular Phylogenetics and Evolution4.066Zhang YX, et al.2012 Junhttp://www.ncbi.nlm.nih.gov/pubmed/22415014
DsbM, a novel disulfide oxidoreductase affects aminoglycoside resistance in Pseudomonas aeruginosa by OxyR-regulated responseJournal of Microbiology1.276Wang X, et al.2012 Dechttp://link.springer.com/article/10.1007/s12275-012-2177-3
Sinomyces: a new genus of anamorphic PleosporaceaeFungal Biology2.082Wang Y, et al.2011 Febhttp://www.ncbi.nlm.nih.gov/pubmed/21315316
Identification of ethylene-responsive genes in ethrel-treated shoot apices of cucumber by suppression subtractive hybridizationPlant Molecular Biology Reporter5.319Jin 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 characterizationMycological Progress1.606Pei YF, et al.2011 Mayhttp://link.springer.com/article/10.1007/s11557-010-0686-1
Molecular and morphological description of two new species of Stemphylium from China and FranceMycologia2.11Wang Y, et al.2010 May-Junhttp://www.ncbi.nlm.nih.gov/pubmed/20524602
Ulocladium cantlous sp. nov. isolated from northwestern China: its morphology and molecular phylogenetic positionMycologia2.11Wang Y, et al.2010 Mar-Aprhttp://www.ncbi.nlm.nih.gov/pubmed/20361504
Molecular characterization, immunohistochemical localization and expression of a ribosomal protein L17 gene from Apis cerana ceranaInsect Biochemistry and Physiology1.515Fei Meng, et al.2010 Octhttp://onlinelibrary.wiley.com/doi/10.1002/arch.20386/abstract
The complete mitochondrial genome of Spilonota lechriaspis Meyrick (Lepidoptera: Tortricidae)Molecular Biology Reports2.506Jin-Liang Zhao, et al.2011 Aughttp://link.springer.com/article/10.1007/s11033-010-0491-6
Halomonas songnenensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soilsIJSEM Juquan Jiang,et al.2014 Mayhttp://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 stressJournal of Insect Physiology2.379Haihong Jia, et al.2014 Janhttp://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 Research1.987Hao-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 ChinaJournal of General Plant Pathology0.893Ai Xin Wang, et al.2014 Janhttp://link.springer.com/article/10.1007/s10327-013-0489-8
MOLECULAR CHARACTERIZATION OF SOLUBLE AND MEMBRANE-BOUND TREHALASES IN THE COTTON MIRID BUG, Apolygus lucorumArchives of Insect Biochemistry and Physiology1.515Yongan Tan, et al.2014 Jun

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

deniedAccessCustomisedMessage=&userIsAuthenticated=false

RNAi mediated knockdown of the ryanodine receptor gene decreases chlorantraniliprole susceptibility in Sogatella furciferaPesticide Biochemistry and Physiology2.111Yao Yang, et al.2014 Janhttp://www.sciencedirect.com/science/article/pii/S0048357513001922
Pollen and seed transmission of Cucumber green mottle mosaic virus in cucumberPlant Pathology2.729H.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 kaempferiPlant Physiology and Biochemistry2.775Qi Yang, et al.2014 Aprhttp://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 striatellusInsect Science1.786Shuang Jia, et al.2014 Apr

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

F45725688822293.f01t01?deniedAccessCustomisedMessage

=&userIsAuthenticated=false

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 one3.7Zhang, YN,et al.2013 Aughttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069715
A novel bifunctional pectinase from Penicilliumoxalicum SX6with separate pectin methylesterase and polygalacturonasecatalytic domainsAppl Microbiol Biotechnol3.689Tao Tu, et al.2014 Janhttp://link.springer.com/article/10.1007/s00253-014-5533-x
Two Origins of Blastemal Progenitors Define Blastemal
Regeneration of Zebrafish Lower Jaw
PloSOne3.54Xuelong Wang, et al.2014 Dechttp://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
PloSOne3.54Yaoming Liu, et al.2014 Novhttp://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-XylosePloSOne3.54Wei Xia, et al.2015 Febhttp://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+ pollutionGeoderma2.772Yun-Bin Jiang, et al.2015 AprJiangsu Provincial Key Laboratory of Materials Cycling and Pollutio

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pEASY®-T3 Cloning Kit

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  • Product Code: CT301
  • Availability: In Stock
  • $255.00

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