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基本描述
| 英文名称 | Ultra-Universal One Step Seamless Cloning Mix | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 储存温度 | -20°C储存,避免反复冻融 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品内容
产品说明
无缝克隆技术是一项简单、高效、快速的DNA克隆技术，可将DNA插入片段定向克隆至任意载体的任意位点。本产品不依赖于T4连接酶，不受截体和目的片段酶切位点的限制，而直接使用同源重组的方法，采用特殊的酶将任意方法线性化后的载体与具有线性化载体两端20-25 bp重叠区域的PCR片段定向重组，50C反应5-15分钟即可快速实现1-5个片段的定向无缝克隆。
产品特点 1.15分钟可以将一个或者多个长、短PCR扩增片段（平/A端） 插入载体。 2.不受载体和插入片段酶切位点的可用性和平端/粘性末端的限制，可以在任意位点 进行克隆。 3.无缝克隆，插入点不会引入不需要的碱基序列。 4.高效、准确，克隆阳性率＞95% 。
自备材料与试剂
·插入片段，特异引物，线性化载体 ·高保真PCR试剂 ·感受态细胞 ·胶回收试剂盒（快速琼脂糖凝胶DNA回收试剂盒） ·PCR仪，PCR反应管等。
线性化载体和插入DNA片段的制备回收
1. 线性化载体的制备回收
（1）
选择合适的克隆位点对载体进行线性化，载体线性化方式有两种：限制性内切酶酶
切消化，反向PCR扩增。
酶切所得线性载体，单酶切或者双酶切均可，酶切后推荐用PCR产物纯化试剂盒 或者胶回收试剂盒纯化载体。因无缝克隆产品体系中没有DNA连接酶，所以不会 引起载体自连，重组产物转化后出现的假阳性克隆 (无插入片段) 是由酶切不完全 未线性化环状载体转化而形成的，所以我们推荐酶切后胶回收纯化，可以将未线 性化载体比例降低到最低程度。 （2）
反向PCR扩增得到线性化载体，推荐使用高保真聚合酶扩增，以减少扩增突变
的引入，使用环状质粒为模板时，建议使用内切酶Dpn I对PCR产物进行消化，以
减少环状质粒模板残留引起的克隆假阳性。如果使用Dpn I消化质粒模板，需80℃
加热20分钟失活Dpn I活性，以避免重组反应时残留Dpn I对克隆载体的降解。
2.插入片段的制备与回收 插入片段的制备可用任意PCR酶扩增，克隆过程不受扩增产物平末端或粘末端（A
尾）的影响（重组过程中将被去除，在最终克隆产物中不会出现），但为了减少扩
增突变，特别是点突变实验中，建议使用高保真聚合酶进行扩增。
一般情况下，PCR产物推荐胶回收纯化以降低背景比例，如果插入片段来源于质粒 模板，且该质粒与重组载体具有相同抗性，需用内切酶Dpn I消化PCR产物，以降 低背景，提高阳性率。 3. 插入片段的引物设计原则 单片段引物设计原则：在插入片段正反向扩增引物的5’端引入线性化载体两末端的 同源序列，使扩增后的插入片段两端分别带有和线性化载体两末端对应的同源序列 （20-25 bp不包含酶切位点） 5’-上游载体末端同源序列（20 bp）+酶切位点（可删除）+基因特异性正向扩增引 物序列（20 bp）-3’ 5’-下游载体末端同源序列（20 bp）+酶切位点（可删除）+基因特异性反向扩增引 物序列（20 bp）-3’ 多片段引物设计原则：载体两端的引物设计原则与单片段克隆时的原则一致，片段 之间设计重叠区域引物。 多片段引物设计原理：片段A的反向引物包含与片段B的正向引物20-25 bp的重叠区 域和特异引物区域，片段B的反向引物包含与片段C的正向引物20-25 bp重叠区域和 特意引物区域，依次类推，两端片段引物包含线性载体两端的同源序列。 如图示：
操作步骤 1. 线性化载体与插入片段的使用量 1.1 载体一般用20-50 ng，插入片段与载体的摩尔比在2:1-3:1之间最佳；多片段连接 .1 的情况下，片段与片段之间摩尔比为1:1。 1.2 单片段克隆用量 最适克隆载体使用量=（0.02 x 克隆载体碱基对数）ng（0.03 pmol） 最适插入片段使用量=（0.04 x 插入片段碱基对数）ng（0.06 pmol） 1.3 多片段克隆用量（2-5个片段） 最适克隆载体使用量=（0.02 x 克隆载体碱基对数）ng（0.03 pmol） 每个插入片段的使用量=（0.02 x 每个插入片段碱基对数）ng（0.03 pmol） 2. 重组反应 注意： 2×Cloning MasterMix含有连接增强剂PEG，很粘稠，从冰箱拿出来温度低时更粘稠，可以 放在手心快速化冻便可降低粘稠度（不影响质量），轻弹混匀。 2.1 按照下表建立反应体系（可使用EP管在室温配制）
3.重组产物转化 3.1 克降感受态置于冰上解冻,取10 uI反应产物加入100 uI感受态细胞中，轻弹管壁 混匀，冰上静置30分钟。 3.2 42CC水浴热激90秒，立即置于冰上2-3分钟。 3.3 加入600 ul无抗LB液体培养基，37C摇床220rpm培养30分钟。3.4 5000rpm离心5分钟，弃多余培养液,剩余100 ul菌液重悬菌体，用无菌涂布棒 均匀涂布在正确抗性的平板上。 3.537'CC培养箱倒置培养12-16小时 4.阳性克隆的鉴定 可根据具体情况，选择菌落PCR监定、提取质粒进行限制性内切酶监定或测序鉴定。
Products content
Product Description Seamless Cloning Technology is a simple, efficient and fast DNA cloning technology that allows targeted cloning of DNA insert fragments into any site of any vector. This product does not depend on T4 ligase, and is not restricted by the truncation and enzymatic sites of the target fragment, but directly uses the homologous recombination method, using a special enzyme to recombine the vector after linearization by any method with the PCR fragment with 20-25 bp overlapping region at both ends of the linearized vector, and the directional and seamless cloning of 1-5 fragments can be quickly realized in 5-15 minutes by 50C reaction.
Product Features 1.15 minutes to insert one or more long or short PCR amplicons (flat/A-end) into the vector. 2. Cloning is not limited by the availability of vector and insert cleavage sites and flat/sticky ends, and can be performed at any site. 3. Seamless cloning, the insertion point does not introduce unwanted base sequences. 4. High efficiency and accuracy, clone positive rate >95%. Prepare your own materials and reagents -Insertion fragments, specific primers, linearized vectors -High-fidelity PCR reagents (Super Pfx MasterMix recommended) -Receptor Cells (DH5α Competent Cell, TOP10 Competent Cell) -Gel Recovery Kit (Rapid Agarose Gel DNA Recovery Kit) -PCR instrument, PCR reaction tubes, etc. Preparation of linearized vectors and insert DNA fragments for recovery 1. Preparation of linearized carriers for recycling (1) Select a suitable cloning site to linearize the vector. There are two ways to linearize the vector: restriction endonuclease digestion and reverse PCR amplification. The linear vector obtained by digestion, single or double digestion is acceptable, after digestion, it is recommended to use PCR product purification kit or gel recovery kit to purify the vector. Since there is no DNA ligase in the Seamless Cloning System, it will not cause the vector to self-associate. False-positive clones (without insert fragments) after recombinant product transformation are formed by incomplete cleavage of non-linearized cyclic vectors. Therefore, we recommend purification by gel recovery after cleavage to minimize the proportion of non-linearized vectors. (2) When the linearized vector is obtained by reverse PCR amplification, it is recommended to use high-fidelity polymerase amplification to reduce the introduction of amplification mutations. When using cyclic plasmid as template, it is recommended to use the endonuclease Dpn I to digest the PCR product to reduce the cloning false positives caused by the residual cyclic plasmid template. If Dpn I is used to digest the plasmid template, it is necessary to inactivate the activity of Dpn I by heating at 80℃ for 20 minutes to avoid the degradation of the cloning vector by residual Dpn I during the recombination reaction. 2. Preparation and recovery of insert fragments The preparation of insert fragments can be amplified by any PCR enzyme, and the cloning process is not affected by the flat or sticky ends (A-tails) of the amplified product (which will be removed during recombination and will not be present in the final cloned product), but in order to minimize the number of amplification mutations, especially in point mutation experiments, it is recommended to use a high fidelity polymerase (Super Pfx Master Mix) for amplification. In general, it is recommended that the PCR product be purified by gel recovery to reduce the proportion of background. If the insert fragment is derived from a plasmid template and the plasmid has the same resistance as the recombinant vector, the PCR product should be digested with the endonuclease Dpn I to reduce the background and increase the positive rate. 3. Principles of primer design for insertion fragments Principle of single fragment primer design: introduce homologous sequences at the 5' end of the primer for forward and reverse amplification of the inserted fragment, so that the amplified inserted fragment carries homologous sequences corresponding to the two ends of the linearized vector (20-25 bp without enzyme cleavage site). 5'-upstream vector terminal homologous sequence (20 bp) + digestion site (can be deleted) + gene-specific forward amplification primer Sequence (20 bp)-3' 5'-downstream vector terminal homologous sequence (20 bp) + digestion site (can be deleted) + gene-specific reverse amplification primer Sequence (20 bp)-3' Principle of multi-fragment primer design: The primer design principle at both ends of the vector is the same as that of single fragment cloning, and the primer for overlapping region between fragments is designed. Principle of multi-fragment primer design: the reverse primer of Fragment A contains a 20-25 bp overlap region with the forward primer of Fragment B and a specific primer region, the reverse primer of Fragment B contains a 20-25 bp overlap region with the forward primer of Fragment C and a specific primer region, and so on, and the primers of the two ends of the fragments contain homologous sequences of the two ends of the linear vectors. As shown:
procedure
1. Amount of linearized vectors and insert fragments used 1.1 Vectors are generally 20-50 ng, with an optimal molar ratio of insert fragments to vector between 2:1 and 3:1; in the case of multiple fragment ligation .1, the fragment-to-fragment molar ratio is 1:1. 1.2 Single fragment cloning usage Optimal cloning vector usage = (0.02 x number of cloning vector base pairs) ng (0.03 pmol) Optimal insertion fragment usage = (0.04 x number of insertion fragment base pairs) ng (0.06 pmol) 1.3 Multi-fragment cloning usage (2-5 fragments) Optimal cloning vector usage = (0.02 x number of cloning vector base pairs) ng (0.03 pmol) Usage per insert fragment = (0.02 x number of base pairs per insert fragment) ng (0.03 pmol) 2. Reorganization reactions Note: The 2×Cloning MasterMix contains PEG, which is very viscous, and even more so when it is taken out of the refrigerator at low temperatures. You can reduce the viscosity by quickly thawing it in the palm of your hand (this does not affect the quality), and then mixing it with a gentle flick. 2.1 Set up the reaction system according to the following table (can be prepared at room temperature using EP tubes)
2.2 Control reaction system (optional) 2.3. Mix gently and react at 50°C in a water bath or PCR instrument for 5-15 minutes, or for ligations of more than 3 fragments, the reaction time can be extended 2.3. to 30 minutes. At the end of the reaction, cool the EP tubes on ice and transform them directly or store them at 2.3. -20℃. 3. Reorganization product transformation 3.1 Grams of the sensory state was thawed on ice, and 10 uI of the reaction product was added to 100 uI of the sensory cell, and the wall of the tube was flicked. Mix well and let stand on ice for 30 minutes. 3.2 Heat-stimulate in a 42 cc water bath for 90 seconds and immediately place on ice for 2-3 minutes. 3.3 Add 600 ul of LB liquid medium without antimicrobial resistance and incubate at 220 rpm for 30 min at 37C. 3.4 Centrifuge at 5000 rpm for 5 min, discard excess culture medium, resuspend the organisms with the remaining 100 ul of culture medium, and use a sterile blotting rod. Spread evenly on the correctly resistant plate. 3.537'CC incubator inverted incubation for 12-16 hours 4. Identification of positive clones Depending on the specific situation, we can choose colony PCR monitoring, plasmid extraction for restriction endonuclease monitoring or sequencing identification. Bring your own instruments Thermostatic mixer. Pre-experiment Preparation and Important Notes 1. Read these instructions carefully before experimenting. 2. If Proteinase K is to be stored for a long period of time, please keep it at -20℃. 3. Check Buffer RLC for crystallization or precipitation prior to use, and if crystallization or precipitation occurs, redissolve Buffer RLC in a 56°C water bath. 4. Pre-treatment of tissue samples: Take 20 mg of tissue samples into 1.5 mL centrifuge tubes (self-provided), add 500 μL of Buffer RLC, and after the tissue homogenizer breaks up, centrifuge the samples for 1 minute at 12,000 rpm (~13,400×g), and take 200 μL of supernatant as samples.
procedure 1. Take a 1.5 mL centrifuge tube (provided), add 500 μL of Buffer RLC, 200 μL of sample, 20 μL of Proteinase K, vortex for 5 s, and then place it in a thermostatic mixer at 1200 rpm for 10 min at room temperature. Note: For wet swab samples, 200 μL of sample was taken after sufficiently shaking and mixing. Note: For wet swab samples, 200 μL was taken from the sample after it was soaked in 400 μL of saline, shaken and mixed thoroughly for 5 minutes, and then centrifuged at 12,000 rpm for 1 minute, and 200 μL was taken for extraction. 2. Instantly remove the centrifuge tube and add the solution from step 1 to the Spin Columns DM in the collection tube. centrifuge at 12,000 rpm (~13,400 x g) for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube. 3. Add 500 μL of Buffer PGWT to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube. 4. Add 500 μL of Buffer GWT2 to the adsorbent column, centrifuge at 12,000 rpm for 1 minute, pour off the waste liquid from the collection tube, and return the column to the collection tube. 5. Centrifuge at 12,000 rpm for 2 minutes and pour off the waste liquid in the collection tube. Place the adsorption column at room temperature for 2 minutes and allow to dry. 6. Place the column in a new collection tube (RNase-Free Centrifuge Tube), add 40-100 μL of RNase-Free Water to the center of the column membrane, let it stand at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the nucleic acid solution. Store at -80℃ for a long time.
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