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查看更多产品信息 MultiSite Gateway® Three-Fragment Vector Construction Kit - FAQs (12537023)
21 个常见问题解答
在BP/LR Clonase反应的一步法实验方案中，不建议用BP Clonase酶和LR Clonase酶替代BP Clonase II 酶/LR Clonase II酶，因为这样的重组效率非常低。
有的，我们能提供针对BP/LR Clonase反应的一步式实验方案DNA可以在一步反应后被克隆到目的载体中，从而节省了您的时间和金钱。
建议使用一个供体载体进行一次BP反应以获得一个入门克隆。然后将这一入门克隆和目的载体进行一次LR反应以获得新的表达克隆。
5X LR Clonase缓冲液或5X BP Clonase缓冲液不作为单独产品出售。它们作为酶试剂盒的一部分进行销售。
我们不提供任何用于在植物内表达的Gateway载体。
抱歉，我们不单独出售任何的MultiSite Gateway相关载体。它们只能作为试剂盒的一部分。
•检查是否使用了正确的Clonase酶以及它是否功能正常（请确保使用LR Clonase II Plus酶）。
•检查是否使用了正确的入门载体和目标载体组合。
•确保使用了正确的入门克隆（以及它们测序正确）。
•检查在反应中是否使用了推荐用量的入门载体和目标载体。
•进行LR反应阳性对照以确定入门克隆是否有问题（请注意：MultiSite GW 3片段试剂盒仅提供单一对照载体pMS/GW，它可用于生成所有对照pENTR载体）。
•检查是否使用了正确的抗生素进行筛选。
•检查目标载体内的att位点序列是否正确。
•延长孵育时间至最长18小时。
标准的MultiSite Gateway反应是在25摄氏度孵育16-18小时。使用2 µL或4 µL（对于4片段重组）反应产物转化One Shot Mach1细胞。使用Mach1细胞替代TOP 10细胞以获得最高效率。对于2片段和3片段重组反应，重要的是保证最优的质粒摩尔比（20 fmole的 pDEST + 10 fmole的pENTR载体）。一般可以获得数以百计的氨苄抗性克隆，克隆效率达到90%（3片段重组反应的效率稍低，通常为70-90%）。对于2片段重组，可以用SOC培养基按1:10比例稀释转化产物，然后涂板50或100 ul。对于3片段重组反应，使用50或100 ul转化产物涂板。对于4片段重组反应，每10 fmole pENTR质粒对应20 fmole的pDEST 载体。孵育16小时并转化Mach1细胞。4片段重组的克隆数和转化效率通常会降低。根据克隆的插入片段不同，得到的正确克隆数量从80%到30%不等。
In the single-step protocol for the BP/LR Clonase reaction, we would not recommend substituting the BP Clonase II/LR Clonase II enzymes with BP Clonase /LR Clonase enzymes as this would result in very low recombination efficiency.
Yes, we have come up with a single-step protocol for BP/LR Clonase reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.
We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.
We do not offer the 5X LR Clonase buffer and 5X BP Clonase buffer as standalone products. They are available as part of the enzyme kits.
We do not offer any Gateway vectors for expression in plants.
We do not offer any of the MultiSite Gateway vectors as standalone products. They are only available as part of the kits.
– Check whether the correct Clonase enzyme was used and whether it was functional (make sure that LR Clonase II Plus was used).
– Check whether the correct combination of Entry vectors and Destination vector was used.
– Ensure that the correct Entry clones were used (and that they were sequenced).
– Check if the recommended amount of Entry vector and Destination vector was used in the reaction.
– Perform the LR reaction positive controls to determine which Entry clone may be faulty (note that MultiSite GW 3-Fragment kit only comes with a single control vector, pMS/GW, that is used to make all control pENTR vectors).
– Check whether the correct antibiotic was used for selection.
– Check whether the att site sequences in the Destination vector are correct.
– Increase the incubation time up to 18 hours.
Standard MultiSite Gateway reactions are incubated for 16-18 hours at 25 degrees C. 2 or 4 µL (for 4 fragment recombination) aliquots are transformed into One Shot Mach1 cells. Use Mach1 cells instead of Top10 cells for the highest efficiency. For 2- and 3-fragment recombination reactions, it is important to keep optimum molar ratios of the plasmids (20 fmoles pDEST + 10 fmoles pENTR vectors). Hundreds of AmpR colonies can be obtained with a cloning efficiency of 90% (expected efficiency is a bit lower for 3-fragment recombination, usually 70-90%). For 2-fragment recombination dilute the reaction 1:10 in SOC medium and plate 50 and 100 µl. For 3-fragment recombination, plate 50 and 100 µL of each transformation. For 4 fragment recombination use 10 fmoles of each pENTR plasmid plus 20 fmoles of the pDEST vector. Incubate for 16 hours and transform into Mach1 cells. The number of colonies and transformation efficiency is normally reduced for 4 fragment recombination. The number of correct clones has been observed to be as high as 80% and as low as 30%, depending on the inserts being cloned.
Detection limits dependend on many factors, including primer design, target size, and the abundance of message. In our hands, this system was able to detect GAPDH mRNA from as little as 1.0 pg of total HeLa RNA when used in conjunction with Platinum Taq DNA Polymerase High Fidelity.
Platinum Taq DNA Polymerase is precomplexed with a mixture of antibodies that inhibit polymerase activity until the initial denaturation step in PCR. As a result, nonspecific polymerase acitivty at lower temperatures during set-up and reverse transcription is eliminated, which provides greater yield and specificity of intended product.
The SuperScript III One-Step RT-PCR System with Platinum Taq DNA Polymerase is recommended for amplifying RNA targets up to 4.5 kb and the SuperScript II One-Step RT-PCR System with Platinum Taq DNA Polymerase is recommended for amplifying RNA targets up to 3.5 kb. Even though the SuperScript II and III One-Step RT-PCR Systems with Platinum Taq High Fidelity can amplify smaller targets, it is recommended for amplifying RNA targets from 1 kb up to 9 kb.
All the following lipids are stable at 4º C for at least one year:
11668-019 Lipofectamine 2000
10362-100 Cellfectin II
10459-014 DMRIE-C
10964-013 Lipofectamine PLUS
18292-011 Lipofectin
18324-012 Lipofectamine
Find additional tips, troubleshooting help, and resources within our Transfection Support Center.
Gateway Cloning Technology is an easy-to-use system for cloning and subcloning DNA segments (e.g. genes of interest), facilitating gene functional analysis, protein expression, and the integration of technology platforms. One can also readily clone PCR products into so-called Gateway "Entry" vectors. To shuttle inserts from one vector to another, the Gateway Cloning Technology uses bacteriophage lambda-based site-specific recombination. There is no need to use restriction enzymes and ligase to subclone inserts.
One advantage of Gateway Cloning Technology is that genes present in a single Gateway Entry vector can be subcloned into multiple different Gateway Destination vectors. After this 1 hour in vitro subcloning reaction, a high percentage of the colonies obtained carry the desired expression clone. For more details, please see the product manual for cat# 12535029 or cat# 12535037.