Baorui Biotech's high-efficiency Seamless cloning kit is now available!

Simple operation

Compared with traditional enzyme digestion ligation, a single enzyme digestion ligation can only link one fragment, while seamless cloning can complete the cloning of one or more fragments in a single reaction.

Compared with enzymatic digestion ligation, seamless cloning does not involve the process of enzymatic digestion treatment. The inserted fragments and linear vectors can be prepared by PCR amplification (or enzymatic digestion), and only the recombinant system needs to be treated at 50 ℃ for 15-60 minutes to be transformed.

Seamless cloning is not limited by restriction endonuclease sites and can insert the target fragment at any position on the vector.

The positive rate of seamless cloning is higher than that of the traditional enzymatic digestion ligation method.

The experimental period is short.

Compared with enzymatic digestion ligation, seamless cloning does not involve the process of enzymatic digestion treatment and has a shorter experimental cycle.

To enhance the ligation efficiency, enzymatic ligation usually requires ligation treatment for 1 hour or even longer. For seamless cloning, only the recombinant system needs to be treated at 50 ℃ for 15 to 60 minutes before conversion can be carried out.

It can be applied in a variety of ways

According to the experimental purpose, seamless cloning can be carried out for rapid cloning, high-throughput cloning, seamless cloning, DNA site-directed mutagenesis and long transcript cloning, etc.

The 2×Seamless Cloning Premix seamless cloning kit can achieve seamless cloning of up to 5 inserted fragments at one time, with a large number of clones and a high positive rate (>95%).

The recombination efficiency of different quantities of fragments

Figure 2 shows the number of monoclones after homologous recombination of different numbers of fragments

Positive rate

Figure 3 shows PCR verification of colonies after homologous recombination of different numbers of fragments

A: Validation of vector and single-fragment recombinant cloning;

B: Verification of recombinant cloning of the vector with 3 fragments;

C: Verification of recombinant cloning of the vector with 4 fragments;

D: Validation of recombinant cloning of the vector with 5 fragments.

There are no or very few converters on the plate

The primer design is incorrectPrimers should contain 15-25 bp homologous arms and have a GC content between 40-60%.

The proportion of the assembly system is inappropriateConfigure according to the recommended dosage in the manual. Adding too much or too little linear carrier and fragment will both have an impact on the recombination effect.

Linear carriers and fragment impurity:Low purity of nucleic acids will affect the recombination effect. The concentration of linear vectors and fragments can be increased.

Low transformation efficiency of competent cellsThe transformation efficiency of competent cells should be greater than 10⁷cfu/μg.

Most clones do not contain insertion fragments or contain incorrect insertion fragments

The PCR products contain non-specific amplification products:Optimize the PCR amplification system to enhance specificity; Recover PCR amplification products from glue and single fragments. Identify more converters.

The amplification template is not processed thoroughlyA negative control was set up during conversion to verify whether the enzymatic digestion or amplification products were completely linearized. Optimize the enzyme digestion system and increase the amount of enzyme used or the digestion time.