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View additional product information for Zero Blunt™ TOPO™ PCR Cloning Kit, with One Shot™ MAX Efficiency DH5α-T1R E. coli - FAQs (K282040, K282020)
41 product FAQs found
两个载体的骨架非常相似。主要的不同在于pCR4-TOPO载体测序引物位点距离PCR产物插入位点仅33 bp。这使得对插入序列进行测序时需要读取的载体序列能最小化，从而使pCR4-TOPO载体非常适合进行测序应用。
两个载体的骨架非常相似。主要的不同在于pCRII-TOPO 载体是一个双启动子载体，包含SP6和T7启动子用于体外转录/测序，而 pCR2.1-TOPO仅包含T7启动子用于体外转录/测序。两个载体均含有M13 正向和反向引物位点用于测序或PCR检测。
对照模板的序列是受专利保护的。
我们建议起始摩尔比为1:1(插入片段：载体)，范围为0.5：1到2：1(插入片段：载体)。对TOPO克隆来说，2kb大小的 PCR产物的ng值应在5- 10ng之间。
计算公式：< br / >
插入片段长度（bp）/ 载体长度（bp）x 载体用量（ng）= 插入片段：载体比为1：1时所需的插入片段的用量（ng）。
您可能需要尝试不同的插入片段:载体比例，范围从1:1至15:1。
公式：
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
(插入片段长度 (bp) X 载体重量(ng) ) / 载体长度 (bp) = 插入片段:载体比例为1:1时所需的插入片段重量(ng)
仅使用载体进行转化不是推荐的阴性对照实验。拓扑异构酶改构过程不是一个效率100%的过程，因此，在您的混合物中将会存在“不含插入片段的载体”，因此会产生克隆。
磷酸化的产物可以进行TA克隆但不能进行TOPO克隆。这是因为所需的磷酸基团已经包含在载体DNA和拓扑异构酶形成的中间体复合物中。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体的线性载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
你可能克隆了一段假阳性序列。TA和TOPO克隆对于存在于某些PCR反应中的小片段（< 100 bp）非常高效。使用硅基DNA纯化系统对插入片段进行凝胶纯化或者电洗脱。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
出现这一问题的原因可能有：
•插入片段没有破坏lacZ基因的读码框。如果你的插入序列较短（< 500 bp），则可能出现浅蓝色克隆。可以分析一些这类克隆，因为它们可能含有插入片段。
•使用了不能在3´ 末端加A的聚合酶。如果您使用的是具有校读活性的聚合酶，例如AccuPrime Pfx 或Platinum Pfx，您将需要进行一个后续Taq酶处理步骤以添加3’ A突出末端。
• PCR产物在连接之前进行了凝胶纯化。凝胶纯化可以去除单个的3’ A突出碱基。如果没有进行凝胶纯化，那么请优化您的PCR反应以便可以直接从PCR产物进入克隆步骤。
• PCR产物在进行连接反应前被存放了很长时间。请使用新鲜PCR产物。即使仅存放1天，连接效率也会下降。
•连接反应中加入了太多扩增反应的成分。PCR反应体系内的高盐成分会抑制连接反应。在连接反应中应使用不超过2-3 μl的PCR反应混合物。
•连接反应中的载体：插入片段摩尔比可能不正确。估计PCR产物的浓度。使连接反应中载体和插入片段的摩尔比大约为1:1或1:3。
通常平板上应该有少量不含插入片段的白色克隆。这些克隆通常比其它克隆大一些，是由于罕见的重组事件会导致质粒上部分序列缺失引起的（通常是从多克隆接头到F1起始点附近的一个位点）。为找到含有插入片段的克隆，最好挑选各种颜色和形态的克隆进行分析。同一插入片段经常会由于插入方向的不同而产生两种完全不同的克隆形态。
无克隆产生的原因可能是出现了下列问题：
•细菌不是感受态细胞。使用包含在One Shot模块内的pUC18载体检查感受态细胞的转化效率。
•平板的抗生素浓度不正确，或者平板过于陈旧。使用100 μg/mL的氨苄青霉素或50 µg/mL的卡那霉素。确保氨苄平板是新鲜的（储存时间小于1个月）。
•产物被磷酸化了（仅针对TOPO克隆而言）。磷酸化的产物可以进行TA-克隆但不能进行TOPO-克隆。这是因为连接所需的磷酸基团已经包含在载体DNA和拓扑异构酶-形成的中间体复合物中了。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
请考虑以下可能原因：
• pH > 9：检查PCR扩增反应的pH值，并使用1 M Tris-HCl, pH 8的缓冲液进行调节。
• PCR产物过多（或过度稀释）：减少PCR产物的用量（或增加其浓度)
• PCR反应延伸不完全：确保PCR反应最后包含一个7到30分钟的延伸步骤。长的PCR产物将需要更长的延伸时间。
•克隆长插入片段（>1 kb）：请尝试以下一个或全部建议：提高插入片段用量。延长 TOPO克隆反应孵育时间。使用硅基DNA纯化系统（如PureLink系统）或者电洗脱法对插入片段进行凝胶纯化。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
•尽管你使用了Taq聚合酶但是PCR产物仍然没有足够的3´ A突出：提高最后的延伸时间以确保所有的3´末端被腺苷化。Taq聚合酶在模板链的A后面再添加一个A时的效率较低。Taq聚合酶在模板链的C后边添加一个A时的效率最高。您可能需要重新设计引物以使它们包含一个5´ G而非一个5´ T。
请尝试以下建议以提高克隆数量。
•向反应中加入6X盐溶液后，提高TOPO反应在室温孵育的时间。
•电转化能显著提高克隆数量；通常能提高10到20倍。但是，如果进行电转化，重要的一点是TOPO反应混合物要包含稀释的盐溶液，或者，为获得最佳结果，在转化前进行沉淀。为获得高电转化效率，建议如下操作： ◦向 6 µL TOPO反应体系中加入100 µL双蒸水并在37度再孵育10分钟。
◦加入10 µL 3 M 醋酸钠，2 µL 20 µg/µL糖原，300 µL 100%乙醇进行沉淀。置于干冰上或–80摄氏度20分钟，4摄氏度最高速离心15分钟。使用800 µL 80%乙醇洗涤沉淀团块，最高速离心10分钟，倒去乙醇，离心1分钟，然后移除剩余乙醇，避免碰到沉淀团块。干燥沉淀团块（空气干燥或真空吸干）。
◦将沉淀重悬于10 µL ddH2O中并根据常用电转步骤使用3.3 µL 重悬的DNA进行电转化。该电转化将产生比使用同样的TOPO-反应体系进行化学转化时多出高达20倍的克隆。加入100 µL ddH2O并孵育10分钟并不是绝对必要，但是它能充分的稀释反应并可能有助于拓扑异构酶的失活，以使得电转化更容易进行。
我们建议您使用我们的TOPO TA cloning kit for sequencing，它包含pCR4 TOPO载体，或者使用我们的Zero Blunt TOPO PCR cloning kit for sequencing,，它包含pCR4Blunt-TOPO载体。
是的，但是您需要使用牛小肠磷酸酶(CIP)对其进行处理以除去5’磷酸基团才能用于TOPO克隆。
TA克隆
这种克隆方法最初是为配合纯Taq聚合酶（天然的、重组的、热启动）使用而设计的；然而，某些高保真Taq酶和Taq酶混合物通常也适合TA克隆。即使Taq与具有校正能力的聚合酶以10:1或15:1的比例，仍可以产生足够的3’ A突出端去做TA克隆。
推荐使用的我公司的聚合酶包括Platinum Taq、Accuprime Taq、Platinum或Accuprime Taq High Fidelity、AmpliTaq、AmpliTaq Gold或AmpliTaq Gold 360等。
平末端克隆
使用Platinum SuperFi DNA聚合酶等具有校对能力的酶。
定向TOPO克隆
Platinum SuperFi DNA聚合酶效果良好。
不，你不能用pCR-Blunt 或pCR-Blunt-TOPO载体克隆使用Taq DNA聚合酶扩增而来的PCR产物。 pCR-Blunt载体被制备为平末端以接受平末端片段。因为Taq DNA聚合酶的末端转移活性，该酶产生的PCR产物带有3’-A突起。为了将这些PCR产物克隆进pCR-Blunt，你需要将它们的末端抹平（这通常是一个低效的过程）。我们的TA Cloning试剂盒或TOPO TA Cloning试剂盒对于克隆Taq酶生成的PCR产物来说是更好的选择。 TA Cloning试剂盒包含带有3’-T突起的线性化载体用于高效连接Taq酶生成的PCR产物，无需额外操作。
1. 是的，TOPO TA Cloning 和Zero Blunt cloning vectors可以用于体外转录。这些载体含有的T7、SP6 或T3启动子是活跃的噬菌体启动子，相应的聚合酶将从这些启动子转录RNA。
2. 这些载体通常不建议用于体外翻译，因为这些载体的启动子下游（3’端）大多含有一个或多个翻译起始信号（ATG）。ATG在多克隆位点之内，这可能会干扰在插入片段中开放阅读框的翻译。
在-80摄氏度下储存是可以的。TOPO载体可以多次反复冻融而不失活，而且载体在-20摄氏度或者-80摄氏度下储存均能保持稳定性。但是需要注意的是载体在室温下会快速失活（约15分钟），这会导致较低的克隆效率（转化后生长的克隆数会降低）。所以在使用过程中要将其放置在冰浴上以防活性降低，用完后快速放回冰箱。
The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.
The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.
The sequence of the control template is proprietary.
We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)
The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.
Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.
You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
There could be a few possibilities for this:
- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.
No colonies may occur due to the following problems:
Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.
Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.
Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.
We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.
Yes, though you will need to treat it with calf intestinal phosphatase (CIP) to get rid of the 5' phosphate group for TOPO Cloning.
TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.
Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.
Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.
For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.
When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.
No you cannot use pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase. The pCR-Blunt vector is prepared with blunt ends to accept blunt-ended fragments. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this enzyme have 3'-A overhangs. In order to clone these products into pCR-Blunt, you would need to polish the ends to make them blunt (which usually is not an efficient process). Our TA Cloning Kits or TOPO TA Cloning kits are a better choice for cloning Taq-generated PCR products. TA Cloning kits include a linearized vector with 3'-T overhangs for efficient ligation of Taq-generated PCR products without additional manipulation.
Assuming that the primer is at a 50 nM final concentration and 50 mM final salt concentration, the melting temperatures are: M13 Forward (-20) Primer = 52.7 and the M13 Reverse Primer = 45.3. For use in the control PCR reaction we recommend using an annealing temperature of 56C.
Yes, the TOPO TA cloning and Zero Blunt cloning vectors can be used for in vitro transcription. The T7, SP6, and T3 promoters contained in these vectors are active phage promoters, and the corresponding polymerases will transcribe RNA from these promoters.
These vectors are not generally recommended for in vitro translation because most of these vectors contain one or more translational initiation signals (ATG) downstream (3’) of the promoters contained in them, within the multiple cloning sites, which may interfere with attempts to translate an open reading frame in the insert.
Typically 200-300 colonies are obtained for the TOPO kits with 100% inserts (20/20). For the Zero Blunt kit that uses standard ligase, typically 600-1000 colonies are obtained with 95-100% inserts.
Phenol red is added to the TOPO vector (pCRII, pCR2.1, and pCR4), mostly to make it more visible in the tube. The color of the solution is generally pink at room temperature, but at a low pH phenol red will turn yellow. We have tested acidic conditions (low pH) for the TOPO ligation reaction and found that the activity is not affected. At a pH 2.0, there was no observed decrease in efficiency. However, if the TOPO vector turns dark red, or is blue after adding the PCR product, then the PCR product buffer is too basic (pH 9). High pH does have a negative effect on the TOPO vectors, and the number of resulting colonies in this case will drop.
Note: The Directional TOPO and Zero Blunt TOPO vectors contain bromophenol blue dye rather than phenol red, which results in a blue-colored solution. Consult the product manual to confirm which dye is added to your vector, or contact Technical Support for more information.
-80°C storage is fine. The TOPO vector can be freeze-thawed several times without loss of activity, and the vector is stable for storage at either -20°C or -80°C. However, note that the vector rapidly ages at room temperature (~15 min), which will result in a lower cloning efficiency (a reduction in the number of colonies following transformation). So during active use it should be kept on ice to prevent degradation, and should be quickly returned to the freezer.
Stop Solution is a reagent included in older versions of all TOPO kits, and still offered in the TOPO XL kits. It is very similar to the current TOPO cloning kit Salt Solution. Instruction manual versions preceding Version J recommended adding the Stop Solution at the end of the 5 minute TOPO reaction. The basic function of the Stop/Salt Solution is to keep the Topoisomerase from re-binding to the plasmid DNA and nicking the DNA. Later studies found that it was actually more effective to add this component during the TOPO cloning reaction versus afterward, and thus the Stop Solution was replaced with the similar but higher-concentration Salt Solution in most kits.