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Golden Gate Assembly Kit为系列产品，包括几种不同Type IIS型限制酶，本产品采用的限制酶为BpiI。该系列产品均基于Golden Gate Assembly原理， 即通过Type IIS限制酶独特的切割特点得到想要的的粘性末端并通过T4 DNA Ligase对其进行连接。尤其擅长组装难以克隆的序列，如重复序列、高GC序列、TAL (transcription activator-like)效应基因、超短序列 (＜100 bp) 等。
Type IIS型限制性内切酶与传统的限制酶不同，它识别非回文序列并在距其识别位点下游一定距离的位置切割DNA，会在识别序列外切割出任意的粘性末端，因此可以定制切割序列。
克隆过程如下：在目的基因切割位点外设计 IIS 型限制酶识别位点，酶切后该识别位点被消除，不会出现在插入片段中，因此插入片段与载体连接后不会被二次切割；载体上含有与目的基因的切割位点互补的粘性末端，可以与之进行连接且不会引入新的序列，从而实现无缝克隆。
基于上述原理的Golden Gate Assembly Kit (BpiI)，包含酶切连接所需要的酶，且以Mix形式出现，加样更便捷，单次反应最高可进行16个片段的连接，充分满足各种实验需求。
实验原理
1. 以单个DNA片段的插入为例：
注1：此处的载体通过酶切得到，需选择带有BpiI酶切位点的载体。除此之外，载体还可以通过PCR获得，引物设计见下文。
注2：插入片段通过PCR方式获得，需将酶切位点通过引物加入片段末端。推荐使用高保真酶扩增，保证扩增产物正确性。
注3：红色的“TTTTTT”为保护碱基示意，可根据不同酶进行调整，推荐使用6个。
注4：上图仅展示了单片段的连接过程，更多片段的连接原理与之一致，只需改变粘性末端的序列即可增加连接片段数。
实验流程
注意事项
1. 克隆效率受插入DNA的类型、数量和大小的影响很大。虽然可以优化实验条件，但部分外源DNA的细胞毒性和胞内重组无法完全避免。
2. 连接反应所需片段通常PCR获得，但也可以通过预克隆或者合成获得。但无论哪种方式，必须保证相邻的片段间的酶切位点方向的正确性，以保证酶切产生的末端可准确连接。
3. 使用Golden Gate Assembly Kit可以轻松组装重复或很短的难克隆序列，但随着克隆片段数的增加，克隆效率会逐渐下降。克隆片段超过10个时，克隆效率显著下降。为保证获得目标连接产物，建议挑取并筛选 20个左右的菌落。
4. 预克隆的DNA片段相较于PCR扩增获得的片段克隆效率更高，尤其是组装超过80%同源性的重复序列时。
5. 推荐使用PCR仪进行热循环反应，并选择正确的反应程序， 以保证反应顺利进行。
引物设计指南
通过PCR引入BpiI的识别序列，识别序列加在引物的5'端，为确保限制酶能稳定结合到DNA双链上并发挥切割作用，需在识别序列末端加上保护碱基。保护碱基的数量和种类不固定，推荐保护碱基为6 bp，可以保证大部分普通酶切。由于切割位点在识别序列下游且可以是任意序列，因此有2种常见的引物设计方法，如下图所示：
引物设计方法1：此种引物设计方法为保护碱基与酶切位点即“TTTTTTCGTCTCNNNNN”均通过引物引入，其他序列则为插入片段上的序列 (且必须大于15 bp)，两部分共同构成引物。
引物设计方法2：此种引物设计方法为保护碱基与识别序列即“TTTTTTCGTCTCN” 通过引物引入，而“NNNN” 则是插入片段的序列。
注1：两种引物设计主要区别在于“NNNN”是否是插入片段中的序列。为保证无缝克隆，若插入片段与载体均通过PCR获得，则“NNNN”必须是片段或者载体 (二选一)中的序列，即片段与载体需用两种不同的引物设计方式获得。
注2：用于连接的粘性末端“NNNN”的序列对连接特异性存在较大影响，虽然理论上存在256种组合，但需排除回文序列。推荐使用以下网址中的工具进行粘性末端设计：https://goldengate.neb.com/。
此外，引物设计还需注意以下几点：
(1) 为保证扩增正确性，通常要求扩增产物＜5 kb，以此为前提设计引物；
(2) 与模板配对的引物部分Tm值应在58~60℃之间，此时扩增效果最好；
(3) 引物内和引物间不能包含互补序列，以避免发夹形成；
(4) 引物的质量对后续连接的影响巨大，即使连接所需的粘性末端只有一个碱基发生突变，也可能导致连接失败，建议选择可靠的基因合成公司。
PCR 注意事项
1. 建议针对每个片段优化PCR条件，以确保单一的PCR产物；
2. 若PCR获得多个条带，必须凝胶纯化目标DNA。否则将导致组装失败或克隆效率大大降低。胶回收DNA 时，尽量减少紫外切胶的时间，或选择蓝光切胶，以减少紫外线对DNA的损伤；
3. 长片段DNA (＞5 kb) 在胶回收时更容易被损伤，因此推荐使用多个小片段进行连接，而不是单个大片段；
4. 使用ddH₂O或10 mM Tris缓冲液 (pH 8.0)洗脱DNA，避免使用TE缓冲液洗脱DNA，以防止对下游连接产生抑制；
5. PCR产物可以未经纯化直接用于连接反应，但一般只限于单片段克隆。多片段的连接仍需对PCR产物进行纯化。
预克隆指南
预克隆即将PCR获得的DNA片段插入到一个中间载体 (预克隆供体载体，通常在3 kb左右)， 然后将其与其 他插入片段一同加入Golden Gate体系中完成最终组装。
预克隆一般通过TOPO克隆完成，将PCR产物通过拓扑异构酶连接到TOPO载体上，载体含有选择基因供我们筛选阳性克隆。预克隆方法并不固定，可根据您的习惯自行选择克隆手段。
以下以TOPO克隆进行预克隆流程示例：
注1：片段与载体中的黑色箭头代表限制酶酶切方向，从识别位点指向切割位点。
注2：ccdB是一种大肠杆菌致死基因，无外源基因插入时致死，插入片段可破坏ccdB基因的表达，从而在转化时仅允许含插入片段的菌落生长。
对于超过80%一致性的重复/同源序列，推荐使用预克隆；
当插入片段数＞5时，建议使用预克隆分步克隆至目标载体。
使用方法
1. 于冰上配制如下反应体系：
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a. Golden Gate Assembly通常不要求负对照。如果需要，可设置不加插入片段的反应体系为负对照。
b. 对于2000 bp的载体，0.05 pmol用量为60 ng，其他长度可根据这个比例自行计算；
c. 摩尔比为片段: 载体=2: 1，所有插入片段之间比例为1: 1；当片段大于载体时，二者用量互换。
注：片段越多，连接效率和阳性率越低；片段或载体过长，连接效率也会下降。
按上述体系配制完成后，涡旋混匀，于PCR仪中进行反应，反应程序根据片段数按下文推荐程序选择。
2. 推荐反应程序：
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注：对于10个以上的片段组装，虽可实现单次成功组装目标片段，但阳性率和菌落数会出现显著下降，为保证实验成功率，对于10+的组装场景，建议分次组装。
可选恒温程序：
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注：恒温反应对于简单场景如单片段反应效率无影响，对于多片段则会随片段增多而出现一定程度效率下降，但对于2~5片段仍可维持70%以上效率，在条件允许时，建议优先使用变温程序。
3. 反应结束后，可以直接用于转化，或者-20℃保存备用。
4. 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45~60 sec，冰浴5 min。加500 μl SOC或LB培养基，37 ℃振荡培养40~60 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。
5. 阳性克隆检测
克隆完成后，需对产物进行筛选鉴定，一般有酶切与PCR两种方法。
酶切：挑取5~20个菌落至1 ml对应抗性的LB培养基中过夜培养，第二天提取质粒，选择合适的限制酶进行酶切，并通过琼脂糖电泳对酶切结果进行分析，筛选得到目标质粒。
对于常规片段连接，5~10个菌落已可以筛选到目标产物，而重复/同源序列或超过10片段的连接，则需要 20个菌落以筛选正确连接产物。
PCR：
(1) 设计合适的检测引物，根据插入片段长度灵活设计扩增产物长度。对于单片段插入，一般要求扩增长度在500 bp~2 kb之间，正向引物与反向引物分别设计在载体与片段上。对于多片段插入，则正反向引物均设计在载体上，扩增插入片段全长。
(2) 挑取5~20个菌落至1 ml对应抗性的LB培养基中过夜培养，将培养产物分别取1 μl至PCR体系中 (30 μl PCR 体系即可满足扩增需求)。
(3) 扩增程序在常规PCR基础上将第一步变性由95℃ 3 min延长至95℃ 10 min，其余步骤保持不变。扩增完成后进行琼脂糖电泳检测扩增产物是否正确。
(4) 选择扩增正确的菌液进行质粒提取，获得目标连接产物。(推荐对正确的连接产物进行进 一 步的测序验证，防止 PCR 扩增时出现错误)。
常见问题与解决办法
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FAQs
1. 单次反应可以组装的片段数量上限是多少？
目前测试的单次最大组装片段数是15，但菌落数和阳性率已降低至很低水平，为确保您的实验成功，建议单次组装不超过5个片段。若多个片段一次反应组装失败，建议分成几步组装，降低单次反应的组装片段数量。
2. 插入片段的长度范围是多少？
目前测试的可插入片段长度范围为20 bp~10 kb，片段越长，克隆效率越低，建议片段加载体的总长度控制在13 kb以下。
3. 可以延长或缩短反应时间吗？
可以。组装单个片段时，42℃，5 min已经足够产生相当多的转化子；组装多个片段时，推荐30~60个循环的反应，可根据连接效果适当调整循环数，但不建议超过60 ，超过此循环数并不会增加连接效果，反而存在非特异性组装的风险。
4. 反应程序中65 ℃，5分钟的孵育步骤目的是什么 ?
65℃条件下T4 DNA连接酶无法进行连接反应，而限制酶仍可消化未被用于组装的质粒，从而降低空白质粒背景。
5. PCR产物是否可以不纯化直接用于组装反应 ?
可以，但一般仅用于单片段组装且加入量不建议超过1μl。未纯化的PCR产物含有DNA聚合酶，可能会补齐 Type IIS限制酶酶切产生的粘性末端导致非特异性连接，同时残留的DNA聚合酶可能和连接酶产生竞争导致连接效率下降；另外PCR产物可能含有非特异性扩增或引物二聚体，同样会导致目标之外的连接产物。
6. Golden Gate Assembly的产物能否作为模板进行后续的PCR？
可以，组装产物存在闭合环状双链 DNA，可以用于后续PCR扩增，同样也可以用于其他DNA扩增技术，如滚环扩增等。
7. 如果插入片段内部含有BsmBI酶切位点如何解决？
需对插入片段中的BsmBI位点进行同义突变；或对序列进行酶切位点分析，更换其他不含酶切位点的Type IIS型限制酶，如Bbsl，Bsal，BspQl。需注意本试剂盒不适用于其他Type IIS型限制酶，仅用于BsmBl。
8. 如何选择合适的Golden Gate Assembly试剂盒？
这取决于插入片段中有哪些Type IIS型限制酶的识别位点。由于内部位点需要通过点突变消除，因此选择基于插入片段中没有或识别位点最少的Type IIS型限制性内切酶的试剂盒。
9. 该试剂盒的性能与使用IIS型限制性内切酶和T4 DNA连接酶普通构建相比如何？
该试剂盒针对Golden Gate Assembly进行了专门的优化，充分满足10片段及以下的连接反应，效率更高更方便。
10. 哪些因素影响Golden Gate Assembly效率？
插入片段数量，片段长度，底物纯度均会影响连接效率。推荐使用10 kb以内的片段进行连接反应，同时使用经纯化过的底物。
11. 预克隆的优势是什么，什么情况下建议进行预克隆？
预克隆的插入片段保存于环状质粒中，相较于PCR产物更稳定， 可实现插入片段的长期储存。当插入片段较多或较长时，建议进行预克隆分步组装，虽然会增加实验周期，但成功率会更高。
The Golden Gate Assembly Kit is a series of products containing multiple Type IIS restriction endonucleases, among which BsmBI is adopted in this kit. All products in this series work based on the Golden Gate Assembly principle: specific sticky ends are generated via unique cleavage characteristics of Type IIS restriction enzymes, and then ligated by T4 DNA Ligase.It is especially suitable for assembling difficult-to-clone sequences, including repetitive sequences, high-GC sequences, TAL (transcription activator-like) effector genes, ultra-short sequences (<100 bp), etc.
Different from traditional restriction enzymes, Type IIS restriction endonucleases recognize non-palindromic sequences and cleave DNA at a certain distance downstream of recognition sites. They produce arbitrary sticky ends outside recognition sequences, enabling customized cleavage sequence design.
The cloning procedure is as follows: Type IIS restriction enzyme recognition sites are designed outside target gene cleavage sites. These recognition sites are removed after digestion and will not remain in inserted fragments, so inserted fragments cannot be recleaved after ligation with vectors. Vectors carry sticky ends complementary to target gene cleavage sites, allowing ligation without introducing extra sequences to achieve seamless cloning.
Based on the above principle, the Golden Gate Assembly Kit (BpiI) integrates all enzymes required for digestion and ligation in a premixed format for convenient operation. It supports ligation of up to 16 fragments in a single reaction, fully meeting diverse experimental requirements.
Experimental Principle
Taking single DNA fragment insertion as an example:
Note 1: The vector is obtained by enzymatic digestion, and vectors with BpiI sites are required. Alternatively, vectors can be prepared by PCR; primer design is described below.
Note 2: Insert fragments are amplified by PCR, and restriction sites are added to fragment ends via primers. High-fidelity DNA polymerase is recommended to ensure accurate amplicons.
Note 3: Red "TTTTTT" indicates protective bases, adjustable for different enzymes; 6 bases are recommended.
Note 4: The figure only shows single-fragment ligation. Multi-fragment assembly follows the same principle, and more fragments can be connected by altering sticky end sequences.
Experimental Procedure
Precautions
1. Cloning efficiency is greatly affected by type, quantity and length of inserted DNA. Although conditions can be optimized, cytotoxicity and intracellular recombination of partial exogenous DNA cannot be completely avoided.
2. Ligation fragments are usually amplified by PCR, and can also be obtained by pre-cloning or gene synthesis. Regardless of sources, correct orientation of restriction sites between adjacent fragments must be guaranteed for accurate sticky-end ligation.
3. The kit easily assembles repetitive or ultra-short difficult-to-clone sequences, but cloning efficiency declines gradually with increasing fragment quantity. Efficiency drops significantly when more than 10 fragments are cloned. It is recommended to pick and screen around 20 colonies to obtain target ligation products.
4. Pre-cloned DNA fragments show higher cloning efficiency than PCR-amplified fragments, especially for repetitive sequences with >80% homology.
5. A thermal cycler is recommended for thermal cycling reactions with proper programs to ensure smooth reactions.
Primer Design Guide
BsmBI recognition sequences are introduced via PCR and added to the 5' end of primers. Protective bases are required at the end of recognition sequences to ensure stable binding and cleavage of restriction enzymes. The number and type of protective bases are flexible, and 6 bp protective bases are recommended for most routine digestions.Since cleavage sites locate downstream of recognition sequences with arbitrary sequences, two common primer design strategies are illustrated below:
Primer Design Method 1: Protective bases and restriction sites (TTTTTTCGTCTCNNNNN) are introduced by primers, while the remaining sequences (>15 bp) originate from inserted fragments. The two parts form complete primers.
Primer Design Method 2: Protective bases and recognition sequences (TTTTTTCGTCTCN) are introduced by primers, and "NNNN" comes from inserted fragment sequences.
Note 1: The core difference between the two methods is whether "NNNN" belongs to inserted fragments. For seamless cloning, if both fragments and vectors are PCR-amplified, "NNNN" must derive from either fragments or vectors, meaning fragments and vectors adopt different primer designs.
Note 2: Sticky-end sequences "NNNN" greatly affect ligation specificity. Although 256 combinations exist theoretically, palindromic sequences should be excluded. Sticky ends can be designed via online tools: https://goldengate.neb.com/
Additional primer design requirements:
(1) Amplicons are generally controlled below 5 kb to guarantee accurate amplification.
(2) The Tm value of primer regions matching templates should be 58–60 ℃ for optimal PCR performance.(3) No complementary sequences are allowed inside or between primers to avoid hairpin structures.
(4) Primer quality critically affects subsequent ligation. A single base mutation in sticky ends may cause ligation failure. Reliable gene synthesis suppliers are highly recommended.
PCR Precautions
1. Optimize PCR conditions for each fragment to obtain single specific amplicons.
2. Gel purification of target DNA is mandatory if multiple PCR bands appear, otherwise assembly fails or cloning efficiency decreases sharply. Minimize UV exposure during gel cutting, or use blue-light gel cutting to reduce DNA damage.
3. Long fragments (>5 kb) are easily damaged during gel extraction. Multi-fragment ligation is preferred over single large-fragment cloning.
4. Elute DNA with ddH₂O or 10 mM Tris buffer (pH 8.0). TE buffer is prohibited to avoid inhibiting downstream ligation.
5. Unpurified PCR products can be used directly for ligation, but only for single-fragment cloning. Purification is required for multi-fragment assembly.
Pre-Cloning Guide
Pre-cloning refers to inserting PCR-amplified DNA fragments into intermediate donor vectors (~3 kb), then assembling them with other fragments in the Golden Gate reaction system.Pre-cloning is commonly performed via TOPO cloning: PCR products are ligated into TOPO vectors by topoisomerase. Vectors contain selection markers for positive clone screening. Other cloning methods are also acceptable according to experimental habits.The following shows the TOPO pre-cloning workflow:
Note 1: Black arrows in fragments and vectors indicate restriction cleavage directions from recognition sites to cutting sites.
Note 2: ccdB is an E. coli lethal gene. It kills bacteria without inserted fragments, while inserts disrupt ccdB expression and allow positive colonies to grow.
Pre-cloning is recommended for repetitive/homologous sequences with >80% identity. Stepwise pre-cloning into target vectors is advised when more than 5 fragments are inserted.
Protocol
1. Prepare reaction systems on ice:
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a. Negative controls are optional for Golden Gate Assembly. A reaction without inserts serves as negative control if required.
b. 60 ng vector for 2000 bp; dosage can be calculated proportionally for other lengths.
c. Molar ratio: Insert : Vector = 2:1; ratio between inserts=1:1. Swap dosages if fragments are longer than vectors.
Note: More fragments lead to lower ligation efficiency and positive rate. Overlong fragments or vectors also reduce efficiency.
Mix thoroughly by vortexing after system preparation, then run reactions in a thermal cycler with programs matched to fragment quantity.
2. Recommended Thermal Programs
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Note: Assembly of >10 fragments can be completed in one reaction, but colony count and positive rate decline significantly. Stepwise assembly is suggested for high success rate.
Optional Isothermal Programs
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Note: Isothermal reactions perform equally well for single-fragment cloning. Efficiency decreases slightly with more fragments, but remains above 70% for 2–5 fragments. Thermal cycling programs are preferred when available.
3. Completed reaction mixtures can be used for transformation directly, or stored at -20 ℃.
4. Recombinant Product Transformation
Add 5–10 μl reaction mixture into 100 μl competent cells, mix gently, and incubate on ice for 30 min. Heat shock at 42 ℃ for 45–60 sec, then ice-bath for 5 min. Add 500 μl SOC or LB medium, incubate at 37 ℃ with shaking (200 rpm) for 40–60 min. Spread bacterial suspension evenly onto antibiotic plates, and incubate inverted at 37 ℃ overnight.
Note 1: Positive rates vary among competent cells. Cells with transformation efficiency >10⁸ CFU/μg are recommended.
Note 2: Colony quantity depends on concentration 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 appear on negative control plates.
5. Positive Clone Identification
Enzymatic digestion and PCR are two routine screening methods.
Enzymatic Digestion: Pick 5–20 colonies and culture overnight in 1 ml resistant LB medium. Extract plasmids, digest with suitable restriction enzymes, and analyze results via agarose electrophoresis to screen target plasmids.5–10 colonies are sufficient for conventional ligation. 20 colonies are required for repetitive/homologous sequences or >10-fragment assembly.
PCR Detection
(1) Design specific detection primers. Amplicons are recommended between 500 bp–2 kb. Forward and reverse primers are located on vectors and inserts for single-fragment cloning, and both on vectors for multi-fragment cloning.
(2) Pick 5–20 colonies and culture overnight. Transfer 1 μl bacterial culture into 30 μl PCR system.
(3) Extend initial denaturation to 95℃ for 10 min (standard: 95℃ 3 min), keep other steps unchanged. Detect amplicons by agarose electrophoresis.
(4) Extract plasmids from positive bacterial cultures. Sequencing verification is highly recommended to avoid PCR errors.
Troubleshooting
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FAQs
1. What is the maximum fragment number per single assembly reaction?
The maximum tested quantity is 15 fragments, with extremely low colony count and positive rate. No more than 5 fragments are recommended for reliable results. Split assembly steps if multi-fragment ligation fails.
2. What is the length range of inserted fragments?
Valid inserts range from 20 bp to 10 kb. Longer fragments have lower efficiency. Total vector-plus-insert length is recommended below 13 kb.
3. Can reaction duration be adjusted?
Yes. 5 min at 42℃ suffices for single-fragment ligation. 30–60 cycles are used for multi-fragment assembly. Do not exceed 60 cycles to avoid non-specific ligation.
4. Why incubate at 65℃ for 5 min?
T4 DNA Ligase is inactivated at 65℃, while restriction enzymes remain active to digest empty plasmids and reduce background clones.
5. Can unpurified PCR products be used directly for assembly?
Yes, but only for single-fragment cloning with ≤1 μl input. Residual DNA polymerase may fill sticky ends and cause non-specific ligation. Primer dimers and non-specific amplicons also interfere with target assembly.
6. Can Golden Gate products act as PCR templates?
Yes. Circular assembled plasmids are suitable for PCR, rolling circle amplification and other DNA amplification reactions.
7. How to handle internal BsmBI sites in inserted fragments?
Perform synonymous mutation on endogenous BsmBI sites, or switch to other Type IIS enzymes including BbsI, BsaI, BspQI. This kit only matches BsmBI and cannot be used with other enzymes.
8. How to select suitable Golden Gate Assembly Kits?
Choose kits according to endogenous Type IIS sites in target sequences. Select enzymes with zero or minimal recognition sites to minimize mutation work.
9. Performance comparison with conventional enzyme-ligase cloning?
This kit is specially optimized for Golden Gate Assembly, with higher efficiency and simpler operation for ≤10-fragment ligation.
10. What factors affect Golden Gate efficiency?
Fragment quantity, length and substrate purity are key influencing factors. Fragments ≤10 kb and purified substrates are highly recommended.
11. Advantages and applications of pre-cloning?
Pre-cloned fragments stored in circular plasmids are more stable than PCR products and suitable for long-term storage. Stepwise pre-cloning improves success rate for long or numerous fragments, despite longer experimental cycles.
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| 批号(Lot Number) | 证书类型 | 货号 |
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分析证书 | G1522813 |
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