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Your present location:HomeBest SellerspEASY®-T5 Zero Cloning Kit / pEASY®-T5 Zero Cloning Kit
  • Product Name: pEASY®-T5 Zero Cloning Kit
  • Catalog Number: CT501
  • 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 Contents& storage Citations & references Related Images Download  
Overview

Description

pEASY ®-T5 Zero Cloning Vector contains a suicide gene. Ligation of PCR fragment disrupts the expression of the gene. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required.

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

•High cloning efficiency. Positive clones up to 100%.

•No blue/white selection needed.

•Suitable for short and large fragment cloning.

•Kanamycin and Ampicillin resistance genes for selection.

•M13 forward primer and M13 reverse primer for sequencing.

•T3 promoter and 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: 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 ®- T5 Zero 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

CT501-01

CT501-02

(20 rxns)

(60 rxns)

 

pEASY ®- T5 Zero 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

Trans 1-T1 Phage Resistant

Chemically Competent Cell

10×100 μl

30×100 μl

 

Citations & references
Literature Journal IF Author Date Link
High-throughput sequencing reveals the disruption of methylation of imprinted gene in induced pluripotent stem cells Cell Research 10.526 Gang Chang,et al. 2013 Dec http://www.nature.com/cr/journal/v24/n3/abs/cr2013173a.html
Enhanced Telomere Rejuvenation in Pluripotent
Cells Reprogrammed via Nuclear Transfer
Relative to Induced Pluripotent Stem Cells
Cell Stem Cell 25.315 Rongrong Le et al. 2014 Jan http://www.sciencedirect.com/science/article/pii/S1934590913004967
Identification and RNA Interference of the Pheromone Biosynthesis Activating Neuropeptide (PBAN) in the Common Cutworm Moth Spodoptera litura (Lepidoptera: Noctuidae) Journal of Economic Entomology 1.506 Qin Lu, et al. 2015 Apr http://jee.oxfordjournals.org/content/early/2015/05/06/jee.tov108.abstract
Genome organization and transcriptional regulation of Adenosine Deaminase Acting on RNA gene 1 (ADAR1) in grass carp (Ctenopharyngodon idella) Developmental & Comparative Immunology 2.815 Zhicheng Sun, et al. 2015 Feb http://www.sciencedirect.com/science/article/pii/S0145305X15000294
Radiation-Induced Epigenetic Bystander Effects Demonstrated in Arabidopsis Thaliana REGULAR ARTICLES   Wei Xu, et al. 2015 May http://www.rrjournal.org/doi/abs/10.1667/RR13909.1
The Combination of Tet1 with Oct4 Generates High-Quality Mouse-Induced Pluripotent Stem Cells Embryonic Stem Cells/Induced Pluripotent Stem Cells   Jiayu Chen, et al. 2015 Feb

http://onlinelibrary.wiley.com/doi/10.1002/stem.1879/abstract;jsessionid=

1789E23F63E1155C43DB3A23F8F7C4FA.f02t04?

deniedAccessCustomisedMessage=&userIsAuthenticated=false

Pike , a rice blast resistance allele consisting of two adjacent NBS–LRR genes, was identified as a novel allele at the Pik locus Molecular Breeding 2.246 Jing Chen, et al. 2015 Apr

http://link.springer.com/article/10.1007/s11032-015-0305-6

Identification and Genome Characterization of the First Sicinivirus Isolate from Chickens in Mainland China by Using Viral Metagenomics Plos one 3.234 Hongzhuan Zhou, et al. 2015 Oct http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139668
Replacement of Oct4 by Tet1 during iPSC Induction Reveals an Important Role of DNA Methylation and Hydroxymethylation in Reprogramming Cell Stem Cell 25.315 Yawei Gao, et al. 2013 Mar http://www.sciencedirect.com/science/article/pii/S193459091300060X
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