CT101

Description

pEASY ®-T1 Cloning Kit is designed for cloning and sequencing Taq-amplified PCR products.

•5 minutes fast ligation of Taq-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 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 ®- T1 Cloning Vector 1 μl

Gently mix well, incubate at room temperature (20°C-37°C) 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 25°C; for some PCR inserts, optimal results can be achieved with higher temperature (up to 37°C).

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 42°C 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 37°C(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 37°C for 30 minutes.

7.Spread 200 μl or all transformants on the pre-warmed plate. Incubate at 37°C overnight.

 

Contents& storage

 

Component

CT101-01

CT101-02

(20 rxns)

(60 rxns)

 

pEASY ®-T1 Cloning Vector (10 ng/μl)

20µl

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)

50µl

150µl

Trans1-T1 Phage Resistant

Chemically Competent Cells

10×100 μl

30×100 μl

 

Citations & references

Write a review

Please login or register to review

pEASY®-T1 Cloning Kit

  • Views: 372
  • Product Code: CT101
  • Availability: In Stock
  • $255.00

Available Options


LiteratureJournalIFAuthorDateLink
Undifferentiated embryonic cell transcription factor-1 (UTF1) inhibits the growth of cervical cancer cells by transactivating p27 Kip1Carcinogenesis5.635Wu X L, Zheng P S.2013 Julhttp://www.ncbi.nlm.nih.gov/pubmed/23536577
Rapid, Simple and Sensitive Detection of Q Fever by Loop-Mediated Isothermal Amplification of the htpAB GenePLoS Neglected Tropical Diseases4.569Pan L, et al.2013 Mayhttp://www.ncbi.nlm.nih.gov/pubmed/23696915
Conversion of upland to paddy field specifically alters the community structure of archaeal ammonia oxidizers in an acid soilBiogeosciences3.754Alam M S, et al.2013http://www.biogeosciences.net/10/5739/2013/bg-10-5739-2013.pdf
Description of Streptomonospora sediminis sp. nov. and Streptomonospora nanhaiensis sp. nov., and reclassification of Nocardiopsis arabia (Hozzein & Goodfellow, 2008) as Streptomonospora arabica comb. nov. and emended description of the genus StreptomonosporaInternational Journal of Systematic and Evolutionary Microbiology2.112Zhang DF, et al.2013 Julhttp://www.ncbi.nlm.nih.gov/pubmed/23847283
Diversity of Wolbachia in Natural Populations of Spider Mites (genus Tetranychus): Evidence for Complex Infection History and Disequilibrium DistributionMicrobial Ecology3.277Zhang Y K, et al.2013 Aprhttp://www.ncbi.nlm.nih.gov/pubmed/23429887
Microbiota associated with the migration and transformation of chlorinated aliphatic hydrocarbons in groundwaterEnvironmental Geochemistry and Health2.076Guan X, et al.2013 Aughttp://www.ncbi.nlm.nih.gov/pubmed/23420483
Cloning and haplotype analysis of TaSTE, which is associated with plant height in bread wheat (Triticum aestivum L.).Molecular Breeding3.251Zhang W, et al.2013 Janhttp://link.springer.com/article/10.1007/s11032-012-9767-y
Target-site basis for resistance to acetolactate synthase inhibitor in Water chickweed (Myosoton aquaticum L.)Pesticide Biochemistry and Physiology2.111Liu W, et al.2013 Sephttp://www.sciencedirect.com/science/article/pii/S0048357513000874
Bromodichloromethane induces cell proliferation in different tissues of male F344 rats by suppression of E-cadherin expression via hypermethylation or transcriptional activation of c-myc and cyclin D1Toxicology Letters3.145Liao J, et al.2013 Aughttp://www.ncbi.nlm.nih.gov/pubmed/24001804
Bioremediation of Cd and carbendazim co-contaminated soil by Cd-hyperaccumulator Sedum alfredii associated with carbendazim-degrading bacterial strainsEnvironmental Science and Pollution Research International2.618Xiao W, et al.2013 Janhttp://www.ncbi.nlm.nih.gov/pubmed/22529002
Substitution mutation induced migration anomaly of a D10S2325 allele on capillary electrophoresisInternational Journal of Legal Medicine2.686Chen PY, et al.2013 Marhttp://www.ncbi.nlm.nih.gov/pubmed/23064616
Molecular Characterization of msp2/p44 of Anaplasma phagocytophilum Isolated from Infected Patients and Haemaphysalis longicornis in Laizhou Bay, Shandong Province, ChinaPLoS One3.73Wang Y, et al.2013 Octhttp://www.ncbi.nlm.nih.gov/pubmed/24167608
Deep Sequencing of Maize Small RNAs Reveals a Diverse Set of MicroRNA in Dry and Imbibed SeedsPLoS One3.73Li D, et al.2013http://www.ncbi.nlm.nih.gov/pubmed/23359822
Construction of a bicistronic vector for the co-expression of two genes in Caenorhabditis elegans using a newly identified IRESBiotechniques2.399Li D, Wang M.2012 Marhttp://www.ncbi.nlm.nih.gov/pubmed/22401550
Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA genePLoS One3.73Meng S, et al.2012http://www.ncbi.nlm.nih.gov/pubmed/23077478
A practicable detection system for genetically modified rice by SERS-barcoded nanosensorsBiosensors and Bioelectronics5.437Chen K, et al.2012 Aprhttp://www.ncbi.nlm.nih.gov/pubmed/22342698
Two mechanisms underlying the loss of p16(Ink4a) function are associated with distinct tumorigenic consequences for WS MEFs escaping from senescenceMechanisms of Aging and Development3.264Wu X, et al.2012 Aughttp://www.ncbi.nlm.nih.gov/pubmed/22813853
Isolation and Identification of Cellulolytic Bacteria from the Gut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)International Journal of Molecular Sciences2.598Huang S, et al.2012http://www.ncbi.nlm.nih.gov/pubmed/22489111
The TetR-type transcriptional repressor RolR from Corynebacterium glutamicum regulates resorcinol catabolism by binding to a unique operator, rolOApplied and Environmental Microbiology3.829Li T, et al.2012 Sephttp://www.ncbi.nlm.nih.gov/pubmed/22706057
Isolation, screening, and optimization of the fermentation conditions of highly cellulolytic bacteria from the hindgut of Holotrichia parallela larvae (Coleoptera: Scarabaeidae)Applied Biochemistry and Biotechnology1.893Sheng P, et al.2012 Mayhttp://www.ncbi.nlm.nih.gov/pubmed/22544686
Phylogenetically diverse cultivable fungal community and polyketide synthase (PKS), non-ribosomal peptide synthase (NRPS) genes associated with the South China Sea spongesMicrobial Ecology3.277Zhou K, et al.2011 Octhttp://www.ncbi.nlm.nih.gov/pubmed/21519913
Rapid, simple, and sensitive detection of Anaplasma phagocytophilum by loop-mediated isothermal amplification of the msp2 geneJournal of Clinical Microbiology4.068Pan L, et al.2011 Dechttp://www.ncbi.nlm.nih.gov/pubmed/21976758
Identification of novel maize miRNAs by measuring the precision of precursor processingBMC Plant Biology4.354Jiao Y, et al.2011 Octhttp://www.ncbi.nlm.nih.gov/pubmed/22014170
Archaeal and bacterial diversity in hot springs on the Tibetan Plateau, ChinaExtremophiles2.203Huang Q, et al.2011 Sephttp://www.ncbi.nlm.nih.gov/pubmed/21695489
Study on Heterotrophic-Autotrophic Denitrification Permeable Reactive Barriers (HAD PRBs) for In Situ Groundwater Remediation