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  • Product Name: pEASY®-Blunt Simple Cloning Kit
  • Catalog Number: CB111
  • Storage: Competent Cell at -70°C for six months; others at -20°C for one year
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Overview

Description

pEASY ®-Blunt Simple Cloning Vector eliminates the multi-cloning sites of pEASY ® -Blunt Cloning Vector. It 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.

•SR primer and M13 forward primer 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 Simple 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

CB111-01 (20 rxns)

CB111-02 (60 rxns)

pEASY ®-Blunt Simple Cloning Vector (10 ng/μl)

20 µl

3×20 µl

Control Template (5 ng/μl)

5µl

5µl

Control Primers (10 μM)

5µl

5µl

M13 Forward Primer (10 μM)

50µl

150µl

M13 Reverse Primer (10 μM)

50 µl

150 µl

SR Primer (10 μM)

50µl

150 µl

Trans1-T1 Phage Resistant Chemically Competent Cell

10×100 μl

30×100μl

 

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
The ammonia oxidizing and denitrifying prokaryotes associated with sponges from different sea areas Microbial Ecology 3.277 Han M, et al. 2013 Aug http://www.ncbi.nlm.nih.gov/pubmed/23435827
Bacterial and archaeal symbionts in the South China Sea sponge Phakellia fusca: community structure, relative abundance, and ammonia-oxidizing populations Marine Biotechnology (NY) 2.739 Han M, et al. 2012 Dec http://www.ncbi.nlm.nih.gov/pubmed/22310803
Low Diversity Bacterial Community and the Trapping Activity of Metabolites from Cultivable Bacteria Species in the Female Reproductive System of the Oriental Fruit Fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae). International Journal of Molecular Sciences 2.598 Shi Z, et al. 2012 http://www.ncbi.nlm.nih.gov/pubmed/22754363
Detection and quantification of cultured marine Alexandrium species by real-time PCR World Journal of Microbiology and Biotechnology 1.262 Zhang F, Li Z. 2012 Dec http://www.ncbi.nlm.nih.gov/pubmed/22864601
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

A novel frame-shift mutation of GLI3 causes non-syndromic and complex digital anomalies in a Chinese family Clinica Chimica Acta 2.85 Cheng F, et al. 2011 May

http://www.ncbi.nlm.nih.gov/pubmed/21320477

Modulation of cell wall synthesis and susceptibility to vancomycin by the two-component system AirSR in Staphylococcus aureus NCTC8325 BMC Microbiology 3.104 Haipeng Sun,et al. 2013 Oct

http://www.biomedcentral.com/1471-2180/13/286/

Metallothionein 2 (SaMT2) from Sedum alfredii Hance Confers Increased Cd Tolerance and Accumulation in Yeast and Tobacco. Plos One 3.534 Jie Zhang, et al. 2014 Sep http://dx.plos.org/10.1371/journal.pone.0102750
RcRR1, a Rosa canina Type-A Response Regulator Gene, Is Involved in Cytokinin-Modulated Rhizoid Organogenesis PLOS ONE 3.73 Bin Gao,et al. 2013 Aug http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072914
Polymorphisms of Chicken TLR3 and 7 in Different Breeds PLOS ONE 3.73 Wenke Ruan 2015 Mar http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119967
The amino acid residues at 102 and 104 in GP5 of porcine reproductive and respiratory syndrome virus regulate viral neutralization susceptibility to the porcine serum neutralizing antibody Virus Research 2.324 Baochao Fan, et al. 2015 Jun

http://www.sciencedirect.com/science/article/pii/S0168170215001495

Cloning and characterization of a novel Nicotiana tabacum ABC transporter involved in shoot branching Physiologia Plantarum 3.138 Xiaodong Xie, et al. 2015 Feb

http://onlinelibrary.wiley.com/doi/10.1111/ppl.12267/abstract?

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Construction and evaluation of a fluorescence-based live attenuated Escherichia coli delivery system for generating oral vaccine candidate Applied Microbiology and Biotechnology 3.337 Wenxin Liu, et al. 2015 May http://link.springer.com/article/10.1007/s00253-014-6332-0
The broad pattern recognition spectrum of the Toll-like receptor in mollusk Zhikong scallop Chlamys farreri Developmental & Comparative Immunology 2.815 Mengqiang Wang, et al. 2015 May

http://www.sciencedirect.com/science/article/pii/S0145305X15001202

Construction and characterization of an infectious cDNA clone of encephalomyocarditis virus from pigs in China Archives of Virology 2.39 Puxian Fang, et al. 2014 Nov

http://link.springer.com/article/10.1007/s00705-014-2290-1

Identification of two antagonists of the scavenger receptor CD36 using a high-throughput screening model Analytical Biochemistry 2.582 Yanni Xu, et al. 2010 May http://www.sciencedirect.com/science/article/pii/S0003269710000874
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