product composition

　　Product introduction

　　The seamless cloning technology based on the principle of recombination, as a new generation of cloning method, does not rely on cumbersome enzyme digestion and ligation steps, and does not require end-filling operations. It is a simple, fast and efficient DNA directional cloning technology. The LightNing™ DNA Assembly Mix Seamless Cloning Kit can complete single-fragment recombination in as little as 5 minutes with a positive rate of over 95%. The cofactors added in Mix can effectively improve the positive rate of clones; the optimized reaction system can tolerate impurities contained in unpurified PCR products to a certain extent. The upgraded version of seamless cloning kit has higher positive rate and better compatibility.

　　Experimental procedure

　　1. Outline of the experimental procedure

　　Design amplification primers with overlapping sequences at the ends of adjacent fragments; prepare the vector; enzyme digestion or inverse PCR amplification; 50°C reaction for 5~15min; transformation competent

　　2. Preparation of Linearized Cloning Vectors

　　Select an appropriate cloning site and linearize the vector. The linearization of the vector can be accomplished by enzyme digestion or inverse PCR amplification.

　　① It is recommended to use LightNing™ fast endonuclease for double-enzyme digestion to completely linearize the vector to reduce the transformation background (false positive clones); if single-enzyme digestion is used for linearization, the digestion time can be appropriately extended to reduce Circular plasmid residues. Note 1: The vector linearized by double digestion does not need to be dephosphorylated, but needs to be dephosphorylated after single digestion; Note 2: After digestion, the fast endonuclease should be inactivated or the target product should be purified before for recombination reactions.

　　② Inverse PCR amplification preparation In order to reduce the introduction of amplification mutations, it is recommended to use high-fidelity PCR Mix for amplification. It is recommended to use a pre-linearized plasmid as a template to reduce the effect of the residual circular plasmid template on the positive rate of clones.

　　3. Insert PCR primer design The 5' end of the PCR primer must contain a 15-25 nt (recommended 18 nt) sequence homologous to the end of its adjacent fragment (insert or vector). If the vector has sticky ends and the 3' end overhangs, the primer design must include the overhang; if the 5' end overhangs, the primer design may or may not include the overhang. Insert fragment forward amplification primer: 5'-upstream vector end homologous sequence + restriction enzyme site (optional) + gene-specific forward amplification sequence - 3' Insert fragment reverse amplification primer: 3'-gene Specific reverse amplification sequence + restriction enzyme cleavage site (optional) + homologous sequence at the end of the downstream vector—5' Note 1: Try to select a region without repetitive sequences and uniform GC content for cloning. When the vector cloning site is upstream and downstream When the GC content in the 25 nt region is 40%~60%, the recombination efficiency is the highest;

　　Note 2: The connecting end sequence of the pUC 19 vector (Ampr ) provided by this kit is as follows:

　　4. It is recommended to use high-fidelity PCR Mix for PCR amplification of inserts to reduce the introduction of amplification mutations. It is recommended to use the purified PCR product for seamless cloning reaction. If the PCR product is identified as a specific amplification product by agarose gel electrophoresis, it can be used directly, but the sample volume should not exceed 20% of the total reaction volume.

　　5. Recombination Reaction

　　① Configure the following reaction system in an ice-water bath:

　　a. Optimum carrier amount (ng) = 0.02 × carrier base pairs, or 0.03 pmol. b. Insert a single fragment, optimal fragment amount (ng) = 0.04 × fragment base pairs.

　　Note 1: If the length of the inserted single fragment is greater than that of the vector, the amount of the vector and the inserted fragment should be interchanged;

　　Note 2: If the length of the insert is less than 200 bp, the insert should use 5 times the amount of vector;

　　Note 3: If the amount calculated by the above formula exceeds the minimum/maximum value, it is recommended to use the minimum/maximum amount directly;

　　Note 4: The positive rate of clones with too long vector fragments and too long insert fragments will decrease. After the reconstitution reaction system is prepared, gently mix the components with a pipette to avoid air bubbles and do not vortex.

　　② Put the reaction system at 50℃ and react for 5~15 min. Note 1: It is recommended to use a PCR instrument with more precise temperature control for the reaction. Insufficient or too long reaction time will reduce the cloning efficiency; Note 2: When the vector backbone is more than 10 kb or the insert fragment is more than 4 kb, it is recommended to extend The reaction time is 15~30 min; Note 3: After the reaction at 50°C is completed, it is recommended to perform a brief centrifugation to collect the reaction solution to the bottom of the tube.

　　③ Put the reaction solution centrifuge tube on ice to cool, then transform or store at -20℃. Note 1: Recombinant products stored at -20°C are recommended to be used within 1 week.

　　6. Recombinant product transformation Take 5~10 μl of the reaction solution, add it to 100 μl of competent cells, slowly mix by pipetting, and place on ice for 30 min. Heat shock at 42°C for 45-60 sec, ice bath for 5 min. Add 500 μl SOC or LB medium and incubate at 37°C with shaking for 40-60 min (200 rpm). The bacterial solution was evenly spread on the plate containing the corresponding antibiotics, and then incubated at 37°C overnight.

　　Note 1: The final clone positive rate of different competent cells will be different, it is recommended to use competent cells with transformation efficiency > 108 CFU/μg;

　　Note 2: The number of colonies depends on the quantity and purity of PCR products and linearized vectors;

　　Note 3: Positive control plates usually grow a large number of white single colonies, while negative control plates only grow few colonies.

　　7. Detection of positive clones: pick a single colony into 10 μl ddH2O and mix well, lyse at 95°C for 10 min, take 1 μl of the lysate as a template for colony PCR identification, or inoculate a single colony into a resistant medium for culture After overnight, the plasmid was extracted and identified by restriction enzyme digestion.

　　Note 1: It is recommended to use at least one universal primer for colony PCR to avoid false positive results;

　　Note 2: The positive results can be further identified by sequencing if necessary.

　　common problem

　　Storage temperature:    -20°C storage

　　Brand:    Three Lions Bio

EZ22046S-LightSpeed系列  DNA Assembly Mix Plus.pdf