基于重组原理的无缝克隆技术，作为新一代的克隆方法，不依赖繁琐的酶切、连接步骤，也不需要末端补平等操作，依据DNA片段与线性化载体末端的15~25 nt 同源序列的重组，可将插入片段克隆至任意载体的任意位点，载体自连背景极低，是一种简单、快速、高效的DNA定向克隆技术。
该试剂盒一次反应可完成单至多个DNA片段的重组，最快仅需5分钟即可完成单片段重组，且阳性率高于 95%。预混液中使用HiFi Taq DNA Ligase，相较于普通的Taq DNA Ligase，拥有更高的保真度，显著提高无缝克隆成功率。同时，预混液中还添加了转化增强剂，大幅提高转化子数量。在保证保真度和转化效率的情况下，该试剂盒进一步优化了组分，使其稳定性显著提高，可以耐受热、氧环境。
产品应用
快速克隆；多片段DNA组装； DNA定点突变。
实验步骤
1. 实验流程概要
2. 线性化克隆载体制备
选择合适的克隆位点，对载体进行线性化，载体的线性化可以通过酶切或反向PCR扩增完成。
(1) 酶切制备
一些限制性内切酶不能有效地消化超螺旋DNA，因此可能会留下不同数量的未切割载体DNA，降低阳性率。推荐使用快速内切酶进行酶切 (单酶切或者双酶切)，使载体线性化完全，以降低转化背景 (未切割的载体转化获得的假阳性克隆)。
注1：经酶切进行线性化的载体无需去磷酸化，推荐使用双酶切。
注2：酶切完成后，建议将内切酶失活或对载体纯化后用于重组反应。
注3载体胶回收时建议长时间电泳与残留的环状质粒区分开后再切胶，以减少假阳性率。
(2) 反向PCR扩增制备
为减少扩增突变的引入，推荐使用高保真PCR Mix进行扩增。推荐使用预线性化质粒作为模板，以减少环状质粒模板残留对克隆阳性率的影响。
注1：PCR产物无非特异性条带时，推荐使用DpnI 1 μl加入50 μl PCR产物中37℃ 1 h，80℃ 20 min消化质粒模板即可用于重组反应；反之建议将PCR产物胶回收后使用。
注 2：多片段克隆时，建议将PCR产物纯化后使用。
3. 插入片段 PCR 引物设计
PCR引物的5'端必须包含与其相邻片段 (插入片段或载体)末端同源的15~25 nt (推荐18 nt)序列。假如载体为粘性末端，且3'端突出，则引物设计必须包含突出部分；若5'端突出，则引物设计可以包含突出部分，也可以不包含。
插入片段正向扩增引物：
5'-上游载体末端同源序列+酶切位点 (可选)+基因特异性正向扩增序列- 3'
插入片段反向扩增引物：
3'-基因特异性反向扩增序列+酶切位点 (可选)+下游载体末端同源序列-5'
注1：尽量选择无重复序列且GC含量均匀的区域进行克隆，当载体克隆位点上下游25 nt区域内GC含量为 40%~60% 时，重组效率最高；
注2：当重组转化子产物>10 kb以上时，推荐按20~25 bp同源臂设计引物。
注 3：本试剂盒所提供的pUC 19载体 (Ampr)连接端序列如下：
注4：包含多个重复序列的片段无法采用无缝克隆的策略进行连接。
4. 插入片段的PCR扩增
推荐使用高保真PCR Mix进行扩增，以减少扩增突变的引入。建议使用纯化后的PCR产物进行无缝克隆反应，若PCR产物经琼脂糖凝胶电泳鉴定为特异性扩增产物，可直接使用，但加样体积不应超过总反应体积的 20%。
5. 重组反应
(1) 于冰水浴中配置以下反应体系：
| 组分 | 反应体系 | 阴性对照 ᶜ | 阳性对照 (如有必要) ᵈ | 快速多片段DNA无缝克隆预混液
| 5 μl | 5 μl | 5 μl | | 线性化载体 ᵃ | 50~200 ng | 50~100 ng | pUC 19 Control Plasmid,
Linearized, 1 μl | | 插入片段 ᵇ | 10~200 ng | - | 500 bp Control,
Fragment, 1 μl | | ddH₂O | To 10 μl | To 10 μl | To 10 μl |
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a. 最适载体用量 (ng)=0.02×载体碱基对数，即0.03 pmol。
b. 插入单片段，最适片段用量 (ng)=0.04×片段碱基对数；插入多片段，每片段最适用量 (ng)=0.02×片段碱基对数。
c. 阴性对照可用来确认线性化载体中是否有环状质粒残留，推荐进行。
d. 阳性对照可用来排除其它实验材料及操作因素的影响。
注1：若插入单片段的长度大于载体，则应互换载体与插入片段用量；
注2：若插入片段的长度小于200 bp，则插入片段应使用5倍载体用量；
注3：若按上述公式计算得到的用量超过最低/最高值，则建议直接按最低/最高用量使用；
注4：载体片段过长、插入片段过长或片段数过多，克隆菌落数及阳性率均会降低。
注5：单点突变按照最适载体用量加入体系，多点突变按照多片段每片段最适用量加入体系。
重组反应体系配制完成后，用移液枪轻轻吸打混匀各组分，避免产生气泡，切勿涡旋。
(2) 将反应体系置于 50℃，反应5~60 min。
注1：推荐使用PCR仪等温控比较精准的仪器进行反应，反应时间不足克隆效率会降低；
注2：插入1~2个片段时，推荐反应时间为15 min；插入3~5个片段时，推荐反应时间为30 min；
注3：当载体骨架在10 kb以上或插入片段在4 kb以上时，建议延长反应时间到30~60 min；
注4：50℃反应完成后，建议进行瞬时离心，将反应液收集至管底。
(3) 将反应液离心管置于冰上冷却，之后进行转化或者储存于-20℃。
注：-20℃储存的重组产物，建议在1周内使用。
6. 重组产物转化
取5~10 μl反应液， 加入到100 μl感受态细胞中， 缓慢吸打混匀，冰上放置30 min。42℃热激60 s，冰浴5 min。加500 μl SOC或LB培养基，37 ℃振荡培养50~60 min (200 rpm)。将菌液均匀涂布在含有对应抗生素的平板上，倒置于37 ℃过夜培养。
注1 ：不同感受态细胞最后的克隆阳性率会有所差别，推荐使用转化效率>10⁸ CFU/μg的感受态细胞；
注2：菌落数取决于PCR产物与线性化载体的数量和纯度；
注3：阳性对照平板通常生长大量白色单菌落，阴性对照平板只生长很少的菌落。
7. 阳性克隆检测
挑取单菌落至10 μl ddH₂O中混匀， 95℃裂解10 min后，取1 μl裂解液作为模板，进行菌落PCR鉴定，或将单菌落接种至抗性培养基中培养过夜后，提取质粒进行酶切鉴定。
阳性对照的阳性克隆检测，菌落PCR引物使用通用引物M13F与M13R，酶切鉴定用HindIII与EcoRI。
注1：菌落PCR时建议至少使用一条通用引物，避免假阳性结果；
注2：必要时可进一步对阳性结果进行测序鉴定。
注3 ：M13F：TGTAAAACGACGGCCAGT
M13R：CAGGAAACAGCTATGAC
常见问题
| 问题描述 | 原因 | 解决方法 | | 转化效率低 | 感受态效率低下 | 感受态细胞的转化效率至少需>10⁷ CFU/μg。可进行简单检测，转化0.1 ng pUC19质粒，生长1000个菌落，估算转化效率为10⁷ CFU/μg。当重组转化子产物>10 kb以上时，推荐使用10⁸ CFU/μg感受态细胞或者适用于大质粒DNA和重组产物转化的感受态细胞。 | | 转化效率低 | DNA片段比例不佳 | 按照说明书推荐的最适用量和比例配制反应体系。载体和插入片段的浓度测定：若线性化载体与插入片段已经过纯化，且经电泳检测条带单一或无 Smear 残留时，可使用超微量核酸蛋白检测仪等基于分光光度法的仪器进行浓度测定，但只有当A260/A280在 1.8~2.0之间时浓度值可信；若线性化载体与插入片段未经过纯化，也可使用琼脂糖电泳测定样品浓度。 | | 转化效率低 | DNA片段纯度不够 | 对载体和插入片段进行胶回收纯化。由于EDTA等金属离子螯合剂会抑制无缝克隆反应，因此纯化产物应溶解于ddH₂O中，切勿使用Tris-EDTA等缓冲液。 | | 转化效率低 | 反应产物过量 | 在转化体系中，无缝克隆反应产物体积不应超过感受态细胞体积的10%。 | | 大量克隆不含插入片段 | 载体线性化不完全 | 酶切制备线性化载体时，提高快速内切酶的使用量，延长反应时间，使用胶回收纯化酶切产物。 | | 大量克隆不含插入片段 | 相同抗性质粒污染 | 以质粒为模板进行插入片段PCR扩增时，使用预线性化质粒作为扩增模板，使用DpnI 甲基化敏感型内切酶对扩增产物进行处理，或对产物进行胶回收纯化。 | | 大量克隆不含插入片段 | 平板抗性不足 | 确保使用正确的抗生素，并使用新鲜制备的抗生素平板。 | | 大量克隆含有不正确插入片段 | 非特异性 PCR扩增产物 | 优化PCR体系，提高扩增特异性，或胶回收纯化PCR产物。 | | 大量克隆含有不正确插入片段 | 多重复序列片段 | 插入片段中含有多个重复序列，推荐使用适合重复序列DNA片段克隆的感受态细胞或者采用酶切连接或GoldenGate方法进行克隆。 |
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Seamless cloning technology based on recombination principle is a new-generation cloning method. It does not rely on tedious enzyme digestion and ligation procedures, nor end blunting operations. By recombination of 15–25 nt homologous sequences at the ends of DNA fragments and linearized vectors, inserts can be cloned into any site of any vector with extremely low vector self-ligation background. It is a simple, rapid and efficient directional DNA cloning technique.
This kit completes recombination of single or multiple DNA fragments in one reaction. Single-fragment recombination can be finished in as little as 5 minutes with a positive rate higher than 95%. HiFi Taq DNA Ligase is adopted in the master mix. Compared with ordinary Taq DNA Ligase, it features higher fidelity and significantly improves seamless cloning success rate. Meanwhile, transformation enhancers are added into the master mix to greatly increase the number of transformants. With guaranteed fidelity and transformation efficiency, the kit components are further optimized for superior stability and tolerance to heat and oxygen environments.
Product Applications
Rapid cloning; multi-fragment DNA assembly; site-directed DNA mutagenesis.
Experimental Procedures
1. Overview of Experimental Workflow
2. Preparation of Linearized Cloning Vector
Select appropriate cloning sites to linearize the vector. Vector linearization can be achieved by enzymatic digestion or inverse PCR amplification.
(1) Enzymatic Digestion Preparation
Some restriction endonucleases cannot efficiently digest supercoiled DNA, which may leave undigested vector DNA and reduce positive rates. Rapid restriction endonucleases are recommended for single or double digestion to ensure complete vector linearization and reduce transformation background (false-positive clones generated by undigested vectors).
Note 1: Vectors linearized by digestion do not require dephosphorylation, and double digestion is preferred.
Note 2: After digestion, inactivate endonucleases or purify vectors before recombination reactions.
Note 3: During vector gel recovery, perform prolonged electrophoresis to distinguish residual circular plasmids before gel cutting to reduce false positives.
(2) Inverse PCR Amplification Preparation
High-fidelity PCR Mix is recommended to avoid amplification mutations. Pre-linearized plasmids are suggested as templates to reduce interference of residual circular plasmids on cloning positive rates.
Note 1: If PCR products have no non-specific bands, add 1 μL DpnI into 50 μL PCR products, incubate at 37 ℃ for 1 h and 80 ℃ for 20 min to digest plasmid templates for direct recombination. Otherwise, purify PCR products by gel extraction.
Note 2: Purify PCR products for multi-fragment cloning.
3. PCR Primer Design for Insert Fragments
The 5' end of PCR primers must contain 15–25 nt (18 nt recommended) homologous sequences matching the ends of adjacent fragments (inserts or vectors). For sticky-ended vectors with 3' overhangs, primers must include the overhang region. For 5' overhangs, primers may or may not contain the overhang sequence.
Forward primer for insert amplification:
5'-Upstream vector homologous sequence + Restriction site (optional) + Gene-specific forward sequence-3'
Reverse primer for insert amplification:
3'-Gene-specific reverse sequence + Restriction site (optional) + Downstream vector homologous sequence-5'
Note 1: Clone regions with few repetitive sequences and uniform GC content. Recombination efficiency peaks when GC content within 25 nt upstream and downstream of vector cloning sites is 40%–60%.
Note 2: For recombinant products larger than 10 kb, design primers with 20–25 bp homologous arms.
Note 3: Ligation terminal sequences of pUC19 vector (Ampᵣ) provided in this kit are as follows:
Note 4: Fragments with multiple repetitive sequences cannot be ligated via seamless cloning.
4. PCR Amplification of Insert Fragments
Use high-fidelity PCR Mix to minimize amplification mutations. Purified PCR products are preferred for seamless cloning. Specific PCR products identified by agarose gel electrophoresis can be used directly, but the loading volume shall not exceed 20% of the total reaction system.
5. Recombination Reaction
(1) Prepare the following reaction system on ice bath:
| Component | Reaction System | Negative Control ᶜ | Positive Control (If Required) ᵈ | | Rapid Multi-fragment DNA Seamless Cloning Premix | 5 μl | 5 μl | 5 μl | | Linearized Vector ᵃ | 50~200 ng | 50~100 ng | pUC 19 Linearized Control Plasmid, 1 μl | | Insert Fragment ᵇ | 10~200 ng | - | 500 bp Control Fragment, 1 μl | | ddH₂O | To 10 μl | To 10 μl | To 10 μl |
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a. Optimal vector dosage (ng) = 0.02 × vector base pairs, equivalent to 0.03 pmol.
b. For single insert: optimal fragment dosage (ng) = 0.04 × fragment base pairs.For multiple inserts: optimal dosage per fragment (ng) = 0.02 × fragment base pairs.
c. Negative control verifies residual circular plasmids in linearized vectors and is highly recommended.
d. Positive control eliminates interference from other experimental materials and operations.
Note 1: Swap vector and insert dosages if single insert is longer than the vector.
Note 2: Use 5× vector dosage for inserts shorter than 200 bp.
Note 3: Adopt minimum or maximum dosage when calculated amount exceeds the range limit.
Note 4: Excessively long vectors, inserts or large fragment numbers reduce colony count and positive rate.
Note 5: Apply optimal vector dosage for single-site mutation; apply multi-fragment dosage per site for multi-site mutations.
After system preparation, gently mix components by pipetting to avoid bubbles. Do not vortex.
(2) Incubate the reaction system at 50 ℃ for 5–60 min.
Note 1: Use accurate temperature-controlled instruments such as PCR instruments. Insufficient incubation reduces cloning efficiency.
Note 2: 15 min for 1–2 fragments; 30 min for 3–5 fragments.
Note 3: Extend incubation to 30–60 min for vector backbones >10 kb or inserts >4 kb.
Note 4: Briefly centrifuge to collect reaction liquid at tube bottom after 50 ℃ incubation.
(3) Cool reaction tubes on ice, then proceed to transformation or store at -20 ℃.
Note: Recombination products stored at -20 ℃ should be used within 1 week.
6. Transformation of Recombinant Products
Add 5–10 μL reaction liquid into 100 μL competent cells, mix gently and incubate on ice for 30 min. Heat shock at 42 ℃ for 60 s, then ice bath for 5 min. Add 500 μL SOC or LB medium, incubate at 37 ℃ with shaking (200 rpm) for 50–60 min. Spread bacterial suspension evenly on antibiotic-containing plates, and incubate inverted at 37 ℃ overnight.
Note 1: Positive rates vary with competent cells. Competent cells with transformation efficiency >10⁸ CFU/μg are recommended.
Note 2: Colony quantity depends on quantity and purity of PCR products and linearized vectors.
Note 3: A large number of white single colonies grow on positive control plates, while few colonies grow on negative control plates.
7. Positive Clone Identification
Pick single colonies and resuspend in 10 μL ddH₂O. Lyse at 95 ℃ for 10 min, use 1 μL lysate as template for colony PCR. Alternatively, inoculate colonies into resistant medium, culture overnight and extract plasmids for restriction digestion identification.
For positive control clone identification: use universal primers M13F/M13R for colony PCR, and HindIII/EcoRI for digestion assay.
Note 1: Use at least one universal primer in colony PCR to avoid false positives.
Note 2: Sequencing identification is available for further verification if necessary.
Note 3:M13F: TGTAAAACGACGGCCAGT
M13R: CAGGAAACAGCTATGAC
Troubleshooting
| Problem Description | Cause | Solution | | Low Transformation Efficiency | Low Competent Cell Efficiency | The transformation efficiency of competent cells shall be at least >10⁷ CFU/μg. A simple verification can be performed: transform 0.1 ng pUC19 plasmid, and 1000 grown colonies indicate an estimated transformation efficiency of 10⁷ CFU/μg. For recombinant products larger than 10 kb, competent cells with 10⁸ CFU/μg efficiency, or specialized competent cells for large plasmid DNA and recombinant product transformation, are recommended. | | Low Transformation Efficiency | Improper Ratio of DNA Fragments | Prepare the reaction system in accordance with the recommended dosage and ratio in the instruction manual. Concentration detection of linearized vectors and inserts: For purified fragments with single clear bands and no smear residue on electrophoresis, spectrophotometric instruments such as NanoDrop can be used for concentration measurement. Concentration results are reliable only when the A260/A280 ratio is between 1.8~2.0. Agarose gel electrophoresis can be used to determine sample concentration for unpurified linearized vectors and inserts. | | Low Transformation Efficiency | Insufficient Purity of DNA Fragments | Perform gel extraction purification on vectors and inserts. Metal ion chelators such as EDTA will inhibit seamless cloning reactions. Dissolve purified products in ddH₂O, and avoid using buffers including Tris-EDTA. | | Low Transformation Efficiency | Excessive Reaction Product | In the transformation system, the volume of seamless cloning reaction mixture shall not exceed 10% of the competent cell volume. | | Most Clones Have No Insert Fragment | Incomplete Vector Linearization | When preparing linearized vectors via enzymatic digestion, increase the dosage of rapid restriction endonuclease, extend the reaction duration, and purify digested products by gel extraction. | | Most Clones Have No Insert Fragment | Contamination of Identical Resistant Plasmids | When amplifying inserts via PCR with plasmid templates, use pre-linearized plasmids as templates. Treat PCR products with DpnI methylation-sensitive endonuclease, or purify amplified fragments by gel extraction. | | Most Clones Have No Insert Fragment | Insufficient Plate Antibiotic Resistance | Ensure the correct antibiotics are used, and adopt freshly prepared antibiotic agar plates. | | Most Clones Contain Incorrect Inserts | Non-specific PCR Amplification Products | Optimize the PCR system to improve amplification specificity, or purify PCR fragments via gel extraction. | | Most Clones Contain Incorrect Inserts | Fragments With Multiple Repetitive Sequences | For inserts containing abundant repetitive sequences, use competent cells specialized for repetitive DNA fragment cloning, or adopt restriction ligation or GoldenGate cloning m |
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ZJ26F0535754
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