pEASY ®- Blunt3 Cloning Vector provides dual EcoR I and dual Not I enzyme digestion sites. It is designed for cloning and sequencing Pfu-amplified PCR products. The cloned insert can be released from a single enzyme digestion.
•5 minutes fast ligation of Pfu-amplified PCR products.
•Ampicillin resistance gene for selection.
•Easy blue/white selection.
•T7 promoter, SP6 promoter, M13 forward and M13 reverse primers for sequencing.
•T7 promoter and SP6 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: 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
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 ®- Blunt3 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).
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 ®-Blunt3 Cloning Vector (10 ng/μl)
Control Template (5 ng/μl)
Control Primers (10 μM)
M13 Forward Primer (10 μM)
M13 Reverse Primer (10 μM)
Trans1-T1 Phage Resistant
Chemically Competent Cell