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Your present location:HomeProductsCloning and Mutagenesis SystemCloning Vectors / pEASY®-T1 Cloning Kit
  • Product Name: pEASY®-T1 Cloning Kit
  • Catalog Number: CT101
  • Date: 2015-12-21
  • Views: 67
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    • Specification: 20 rxns 60 rxns
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Overview

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
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