Molecular Cloning

Product description This kit is a new generation homologous recombinant cloning kit. The carefully optimized 2nd generation 2 × Clone Enzyme Premix combines the recombinant enzyme and the buffer required for the recombinant reaction with the addition of a unique recombinant enhancer to significantly improve the efficiency of recombinant cloning. The kit can be directed to clone PCR products to any site of any vector, compatible with unpurified PCR products, directly recovered PCR products, low concentration of rubber recovered products, this product can reassemble the homologous arm GC content of 30%-70% of the joint fragment. The vector was completely linearized, and homologous sequences of the end of the linearized vector of 15-25 bp were introduced into the 5 'end of the positive and reverse PCR primers of the inserted fragment, so that the 5' and 3 'ends of the PCR products of the inserted fragment had exactly the same sequence corresponding to the two ends of the linearized vector, respectively. Under the action of recombinant enzyme, the recombinant reaction of PCR product and linearized vector can be completed in as little as 5 min at 50℃. The positive rate of cloning can reach more than 95%. Components Components No. Name N132081S (20 T) N132081M (50 T) N132081-A 2 × Clone Enzyme Premix 200 μL 500 μL N132081-B 500 bp control insert (25 ng/μL) 5 μL 5 μL N132081-C pUC 19 control vector, linearized (50 ng/μL) 5 μL 5 μL Shipping and Storage Dry ice shipping. -15℃ ~ -25℃ storage, valid for one year. Notes 1. Materials to be prepared by oneself: 1) Sample: Prepare the linearized vector and insert fragments by oneself. 2) Self-prepared reagents (only some of them are listed) : a. super receptive cells: conversion efficiency > 108 cfu/μg, such as DH5α Fast Chemically Competent Cell. b. High fidelity enzyme: 2×High-Fidelity Fast PCR Master Mix (With Dye)（Cat#N132016） or other equivalent. c. Colony PCR mix: 2×HotStart Fast PCR Master Mix (With Dye)（Cat#N132001）or other equivalent product. d. Nucleic Acid dye: Nucleic Acid Gel Stain (10,000× in Water)（Cat#N132109） or other equivalent products. 3) Self-supplied instrument consumables (only listed part): PCR instrument, horizontal electrophoresis tank, glue cutting instrument, EP tube, etc. 2. For your safety and health, please wear lab coats and disposable gloves for operation. 3. This product is for research use ONLY! Instructions 1. Preparation of target vector and insert fragment 1) Preparation of linearized vector Select the appropriate cloning site and linearize the vector. This product is not affected by GC content in the homologous arm region. Linearized vectors can be obtained by restriction enzyme digestion or reverse PCR amplification. a. Enzyme digestion preparation of linearized vector * Double enzyme digestion linearization: complete linearization, low conversion background. Recommended. Single digestion linearization: the degree of linearization is poor. The time of enzyme digestion can be extended appropriately to reduce the conversion background. 【Note】:* False-positive clones without inserted fragments may be formed by the transformation of incomplete linearized circular vectors. If the proportion of such false-positive clones is high, it is recommended to prepare the linearized vectors again and cut the glue for recovery. b. Linearized vector was prepared by reverse PCR amplification Vector amplification using a high-fidelity polymerase (such as 2×High-Fidelity Fast PCR Master Mix (With Dye), Cat#N132016) is recommended to reduce the introduction of amplification mutations. The PCR amplification template should use pre-linearized plasmids as much as possible to prevent the influence of residual ring plasmids on the positive rate of cloning. The recombinant reaction system is compatible with almost all enzyme digestion reaction systems and conventional PCR reaction systems. When the purity of vector enzyme digestion products or reverse PCR amplified products is high, the recombinant reaction can be directly carried out without purification. However, when the purity is low and may contain unlinearized ring plasmids, it is recommended to use a high-quality kit for rubber recovery and purification of the linearized vector to improve the purity of the product and remove part of the unlinearized ring vector, which is conducive to improving the recombinant efficiency. 2) Primer design for a single insert fragment Primer design method: The 5 'and 3' ends of the PCR products of the inserted fragment have identical sequences corresponding to the two ends of the linearized vector, respectively, by introducing 15-25 bp homologous sequences of the 5 'ends of the positive and reverse PCR primers of the inserted fragment. l Insertion fragment forward amplification primer design method: 5 '-upstream vector end homologous sequence + enzyme restriction site (can be retained or deleted) + gene specific forward amplification primer sequence -3' l Insertion fragment reverse amplification primer design method: 3 '- gene-specific reverse amplification primer sequence + cleavage site (can be retained or deleted) + downstream vector terminal homologous sequence -5'   It is recommended to use mature cloning software for primer design. The final primer length is more than 40 bp, and PAGE purification method is recommended for primer synthesis. When calculating the annealing temperature of the amplification primer, only the Tm value of the gene-specific amplification sequence should be calculated, and the homologous sequence at the end of the vector should not be involved in the calculation. In order to obtain high efficiency cloning, it is recommended that Tm≥48℃. 3) Primer design for multiple insertion fragments Primer design method: The 5 'and 3' ends of the PCR products of the inserted fragment have identical sequences corresponding to the two ends of the linearized vector, respectively, by introducing 15-25 bp homologous sequences of the 5 'ends of the positive and reverse PCR primers of the inserted fragment. It is recommended to use mature cloning software to automatically generate insert fragment amplification primer. For manual design, refer to the following example. Take the primer design of three gene segments inserted between the EcoR I and Hind III cleavage sites of pUC18 vector as an example, the specific design scheme of the primer is as follows: a. First design the forward amplification primers of the first fragment and the reverse amplification primers of the third fragment (two inserted fragments adjacent to the vector). l First fragment forward amplification primer design: 5 '-upstream vector terminal homologous sequence + enzyme restriction site (can be retained or deleted) + first fragment gene specific forward amplification sequence -3' l Third fragment reverse amplification primer design method: 3 '-third fragment gene specific reverse amplification sequence + enzyme cut site (can be retained or deleted) + downstream vector terminal homologous sequence -5' b. Secondly, the reverse amplification primers of the first fragment and the forward amplification primers of the second fragment were designed. Homologous sequences for recombination between fragments may be added to the reverse amplification primer of the anterior fragment, to the forward amplification primer of the posterior fragment, or to a part of each fragment. Take the example of adding a homologous sequence to a reverse amplification primer of the front fragment (the first fragment) : l First fragment reverse amplification primer design: 3 '-first fragment gene-specific reverse amplification sequence + second fragment 5' terminal homologous sequence -5 ' l Second fragment forward amplification primer design method: 5 '-second fragment gene specific forward amplification sequence -3' c. Finally, the reverse amplification primers of the second fragment and the forward amplification primers of the third fragment were designed. The design method is consistent with the reverse amplification primers of the first fragment and the forward amplification primers of the second fragment. 4) PCR amplification of the inserted fragment The insertion fragment amplification can be amplified by any PCR enzyme, regardless of whether there is an A-tail at the end of the product (which will be removed during the recombination process and will not be present in the final vector). However, in order to reduce the introduction of amplification mutations, it is recommended to amplify With a high-fidelity polymerase (e.g. 2×High-Fidelity Fast PCR Master Mix (With Dye)(Cat#N132016). It is recommended to use the mature cloning software to automatically generate insert fragment amplification primer. After PCR amplification, a small amount of the product was taken for agarose gel electrophoresis to test the amplification yield and specificity. The recombinant reaction system is compatible with the conventional PCR reaction system. Therefore, if the amplification template is not a circular plasmid with the same resistance as the vector, and the electrophoresis band of the PCR product is single, the amplified product can be directly used in the recombination reaction without purification. However, when the purity of PCR amplification products is low, it is recommended to use high quality kit for rubber recovery and purification of PCR amplification products to improve the purity of products and improve the efficiency of recombinant. 2. Concentration measurement The method of band brightness comparison by agarose gel electrophoresis is recommended for DNA quantification. The linearized vector and the inserted fragment amplification product were subjected to several equal volume dilution gradients. The original product and the diluted product were each taken 1 μL for agar-gel electrophoresis. The band brightness was compared with DNA Marker to determine the approximate concentration (especially in the case of unpurified linearized vector and inserted fragment amplification product). Nucleic acid concentration meter can also be used to determine the linearized vector and the inserted fragment, record the concentration value (ng/μL) and OD260/OD280 values, and then calculate the amount of input.

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