transbionovo

+86-10-57815027      
中文版 | ENGLISH

Your present location:HomeProductsCloning and Mutagenesis SystemCloning Vectors / pEASY®-Blunt Cloning Kit
  • Product Name: pEASY®-Blunt Cloning Kit
  • Catalog Number: CB101
  • Storage: Competent Cell at -70°C for six months; others at -20°C for one year
  • 币种:  
    • Specification: 20 rxns 60 rxns
  • Quantity:  

Quick purchase Purchase Favorite

 

Overview Contents& storage Citations & references Related Images Download  
Overview

Description

pEASY ®-Blunt Cloning Kit is designed for cloning and sequencing Pfu-amplified PCR products.

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

•Kanamycin and Ampicillin resistance genes for selection.

•Easy blue/white selection.

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

•T7 promoter for in vitro transcription.

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

 

Preparation

1.Primer requirement: primer cannot be phosphorylated

2.PCR Enzyme: Pfu 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, 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 ®- Blunt 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

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

CB101-01

CB101-02

(20 rxns)

(60 rxns)

 

pEASY ®- Blunt 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
Phylogenetically diverse endozoic fungi in the South China Sea sponges and their potential in synthesizing bioactive natural products suggested by PKS gene and cytotoxic activity analysis Fungal Diversity 5.319 Yu Z, et al. 2013 Jan http://link.springer.com/article/10.1007/s13225-012-0192-7
Phylogenetic Diversity of Actinobacteria Associated with Soft Coral Alcyonium gracllimum and Stony Coral Tubastraea coccinea in the East China Sea Microbial Ecology 3.277 Yang S, et al. 2013 Jul http://www.ncbi.nlm.nih.gov/pubmed/23503990
Phylogenetically Diverse Denitrifying and Ammonia-Oxidizing Bacteria in Corals Alcyonium gracillimum and Tubastraea coccinea Marine Biotechnology (NY) 2.739 Yang S, et al. 2013 Oct http://www.ncbi.nlm.nih.gov/pubmed/23564007
Spatial distribution of prokaryotic symbionts and ammoxidation, denitrifier bacteria in marine sponge Astrosclera willeyana Scientific Reports 2.927 Yang Z, Li Z. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22829982
Identification of a novel FRMD7 splice variant and functional analysis of two FRMD7 transcripts during human NT2 cell differentiation Molecular Vision 1.987 Li Y, et al. 2011 http://www.ncbi.nlm.nih.gov/pubmed/22128244
Phylogenetically diverse cultivable fungal community and polyketide synthase (PKS), non-ribosomal peptide synthase (NRPS) genes associated with the South China Sea sponges Microbial Ecology 3.277 Zhou K, et al. 2011 Oct

http://www.ncbi.nlm.nih.gov/pubmed/21519913

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system PLoS One 3.73 Jia S, et al. 2011

http://www.ncbi.nlm.nih.gov/pubmed/22039483

Identification and functional analysis of the genes encoding Δ6-desaturase from Ribes nigrum† Journal of Experimental Botany 5.242 Li-Ying Song, et al. 2010 Jun

http://www.ncbi.nlm.nih.gov/pubmed/?term=Identification+and

+functional+analysis+of+the+genes+encoding+%CE%946-

desaturase+from+Ribes+nigrum%E2%80%A0

The Distribution and Common Amino Acid Polymorphisms of Human Papillomavirus (HPV)-31 Variants in 2700 Women from Northern China PLoS One 3.73 Mengfei Liu, et al. 2014 Jun http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0099141
Family-Wide Survey of miR169s and NF-YAs and Their Expression Profiles Response to Abiotic Stress in Maize Roots PLoS One 3.73 Mingda Luan, et al. 2014 Mar http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0091369
A new porcine reproductive and respiratory syndrome virus strain with highly conserved molecular characteristics in its parental and attenuated strains Virus Genes 1.769 Xing Liu, et al. 2014 May http://link.springer.com/article/10.1007/s11262-014-1086-4
The last half-repeat of transcription activator-like effector (TALE) is dispensable and thereby TALE-based technology can be simplified Molecular Plant Pathology 3.877 Chong-Ke Zheng, et al. 2014 Apr

http://onlinelibrary.wiley.com/doi/10.1111/mpp.12125/abstract?

deniedAccessCustomisedMessage=&userIsAuthenticated=false

Three homologous genes encoding functional ∆8-sphingolipid desaturase in Populus tomentosa Genes&Genomics 0.497 Shu-Fen Li, et al. 2014 Jun http://link.springer.com/article/10.1007/s13258-013-0167-4
Comprehensive analyses of molecular phylogeny and main alkaloids for Coptis (Ranunculaceae) species identification Biochemical Systematics and Ecology 1.153 Yang He, et al. 2014 Oct http://www.sciencedirect.com/science/article/pii/S0305197814001458
Novel NAC Transcription Factor TaNAC67 Confers Enhanced Multi-Abiotic Stress Tolerances in Arabidopsis PLoS One 3.73 Xinguo Mao, et al. 2014 Jan

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0084359

Cloning and characterization of goose interleukin-17A cDNA Research in Veterinary Science 1.774 Shuangshi Wei, et al. 2014 Feb

http://www.sciencedirect.com/science/article/pii/S0034528813003251

A novel self-cleavage system for production of soluble recombinant protein in Escherichia coli Protein Expression and Purification 1.429 Yufei Feng, et al. 2014 Jul http://www.sciencedirect.com/science/article/pii/S1046592814000795
Development of a TaqMan-based real-time reverse transcription polymerase chain reaction assay for the detection of encephalomyocarditis virus Journal of Virological Methods 1.9 Wanzhe Yuan, et al. 2014 Jul http://www.sciencedirect.com/science/article/pii/S0166093414002237
The Arabidopsis transcription factor IIB-related protein BRP4 is involved in the regulation of mitotic cell-cycle progression during male gametogenesis Journal of Experimental Botany 5.242 Zhixiang Qin, et al. 2014 Apr http://jxb.oxfordjournals.org/content/65/9/2521.short
The complete mitochondrial genome sequence of Sogatella furcifera (Horváth) and a comparative mitogenomic analysis of three predominant rice planthoppers Gene 2.196 Kai-Jun Zhang, et al. 2014 Jan http://www.sciencedirect.com/science/article/pii/S0378111913013516
Production of free monounsaturated fatty acids by metabolically engineered Escherichia coli Biotechnology for Biofuels 5.552 Yujin Cao, et al. 2014 http://www.biomedcentral.com/content/pdf/1754-6834-7-59.pdf
Targeted genome modification of crop plants using a CRISPR-Cas system Nature Biotechnology 32.44 Qiwei Shan,et al. 2013 Aug http://www.nature.com/nbt/journal/v31/n8/full/nbt.2650.html
ArtR, a novel sRNA of Staphylococcus aureus, regulates α-toxin expression by targeting the 5' UTR of sarT mRNA Med Microbiol Immunol 3.545 Ting Xue,et al. 2013 Aug http://link.springer.com/article/10.1007/s00430-013-0307-0
Methylthioadenosine/S-adenosylhomocysteine nucleosidase (Pfs) of Staphylococcus aureus is essential for the virulence independent of LuxS/AI-2 system International Journal of Medical Microbiology 4.537 Yan Bao, et al. 2013 Mar

http://www.sciencedirect.com/science/article/pii/S1438422113000465

Staphylococcus aureus autoinducer-2 quorum sensing decreases biofilm formation in an icaR-dependent manner BMC Microbiology 3.104 Yu Dan,et al. 2012 Dec

http://www.biomedcentral.com/1471-2180/12/288/

LuxS/AI-2 system is involved in antibiotic susceptibility and autolysis in Staphylococcus aureus NCTC 8325 International Journal of Antimicrobial Agents 4.415 Ting Xue, Liping Zhao,et al. 2012 Aug http://www.sciencedirect.com/science/article/pii/S0924857912003664
Efficient genome editing in plants using a CRISPR/Cas system Cell Research 10.526 Zhengyan Feng, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790235/
D14–SCFD3-dependent degradation of D53 regulates strigolactone signaling Nature 38.597 Feng Zhou,et al. 2013 Dec http://www.nature.com/nature/journal/v504/n7480/abs/nature12878.html
Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew Nature Biotechnology 39.08 Yanpeng Wang, et al. 2014 Jul http://www.nature.com/nbt/journal/v32/n9/full/nbt.2969.html
Identification and characterization of promoters specifically and strongly expressed in maize embryos Plant biotechnology journal 5.67 Xiaoqing Liu, et al. 2014 Sep http://onlinelibrary.wiley.com/doi/10.1111/pbi.12227/full
Meyerowitz, E.M. and Jiao, Y. (2014) The stem cell niche in leaf axils is established by auxin and cytokinin in Arabidopsis. Plant Cell 9.251 Ying Wang, et al. 2014 May http://www.plantcell.org/content/26/5/2055.short
The Chromatin-Remodeling Factor PICKLE Integrates Brassinosteroid and Gibberellin Signaling during Skotomorphogenic Growth in Arabidopsis PLANT CELL 10.656 Dong Zhang, et al. 2014 Jun http://www.plantcell.org/content/26/6/2472.short
Exploring the Catalytic Promiscuity of a New Glycosyltransferase from Carthamus tinctorius Organic Letters 6.2 Kebo Xie, et al. 2014 Sep

http://pubs.acs.org/doi/abs/10.1021/ol502380p

Pi64, Encoding a Novel CC-NBS-LRR Protein, Confers Resistance to Leaf and Neck Blast in Rice Molecular Plant-Microbe Interactions 4.46 Jian Ma, et al. 2015 May

http://apsjournals.apsnet.org/doi/abs/10.1094/MPMI-11-14-0367-R

Analysis of murine B-cell epitopes on bluetongue virus 12 nonstructural protein 1     Wang HaiXiu, et al. 2015 May http://link.springer.com/article/10.1007/s00253-014-6150-4
Biochemical characterization and functional analysis of Udp-glucose dehydrogenase, in the synthesis of biopolymer Ss from Sphingomonas sanxanigenens NX02 Applied Biochemistry and Microbiology 0.735 M. -M. Wu, et al. 2014 Dec http://link.springer.com/article/10.1134/S0003683815010160
Identification and co-localization of perforin-like (TgPLP1) protein in Toxoplasma gondii bradyzoites Experimental Parasitology 1.638 Dan Shan, et al. 2015 Mar http://www.sciencedirect.com/science/article/pii/S0014489415000363
Production of optically pure l-phenyllactic acid by using engineered Escherichia coli coexpressing l-lactate dehydrogenase and formate dehydrogenase Journal of Biotechnology 2.871 Zhaojuan Zheng, et al. 2015 May http://www.sciencedirect.com/science/article/pii/S0168165615002461
Cloning and functional expression of a chitinase cDNA from the apple leaf miner moth Lithocolletis ringoniella Biochemistry (Moscow) 1.303 Xiao-Jun Fan, et al. 2015 Feb

http://link.springer.com/article/10.1134/S000629791502011X

CRKL overexpression suppresses in vitro proliferation, invasion and migration of murine hepatocarcinoma Hca-P cells Biomedicine & Pharmacotherapy 2.023 Qiuyue Lin, et al. 2014 Nov

http://www.sciencedirect.com/science/article/pii/S0753332214001541

Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system Plant Cell Reports 3.071 Plant Cell Reports, et al. 2015 Jun http://link.springer.com/article/10.1007/s00299-015-1816-7
Testing the Effect of Internal Genes Derived from a Wild-Bird-Origin H9N2 Influenza A Virus on the Pathogenicity of an A/H7N9 Virus Cell Reports 8.358 Wen Su, et al. 2015 Aug http://dx.doi.org/10.1016/j.celrep.2015.08.029
Cloning and characterization of TaSnRK2.3, a novel SnRK2 gene in common wheat Journal of Experimental Botany 5.364 Shanjun Tian,et al. 2013 Mar 公司奖励
Related Images

  To be closed automatically in X seconds

View my favorites

Related products
no such related products!