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.
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
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).
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.
pEASY ®- T5 Zero Cloning Vector (10 ng/μl)
Control Template (5 ng/μl)
Control Primers (10 μM)
M13 Forward Primer (10 μM)
M13 Reverse Primer (10 μM)
Trans 1-T1 Phage Resistant
Chemically Competent Cell
|Citations & references|
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|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|
|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|
|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|