CB501

Description

pEASY ®-Blunt 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 selection is not required.

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

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

•No blue/white selection needed.

•Suitable for larger 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: 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 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

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

CB501-01

CB501-02

(20 rxns)

(60 rxns)

 

pEASY ®-Blunt 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 Cells

10×100 μl

30×100 μl

 

Citations & references
LiteratureJournalIFAuthorDateLink
Molecular cloning and characterization of the mitogen-activated protein kinase kinase gene (MKK4) and its promoter sequence from oilseed rape (Brassica campestris L.).Plant Cell, Tissue and Organ Culture (PCTOC)3.633Zhang T G, et al.2013 Aughttp://link.springer.com/article/10.1007/s11240-013-0366-3
Expression, purification and renaturation of truncated human integrin β1 from inclusion bodies of Escherichia coliProtein Expression and Purification1.695Tonglin Shi, et al.2014 Novhttp://www.sciencedirect.com/science/article/pii/S104659281400268X
The Different Potential of Sponge Bacterial Symbionts in N2 Release Indicated by the Phylogenetic Diversity and Abundance Analyses of Denitrification Genes, nirK and nosZPLoS One3.73Zhang X, et al.2013 Junhttp://www.ncbi.nlm.nih.gov/pubmed/23762300


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pEASY®-Blunt Zero Cloning Kit

  • Views: 276
  • Product Code: CB501
  • Availability: In Stock
  • $325.00

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