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查看更多产品信息 MultiSite Gateway® Pro 2.0, for cloning two DNA fragments into a Gateway® destination vector - FAQs (12537102)
22 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。
试剂盒内提供了pDONR 221作为BP重组反应的阳性对照，但是它不能用于生成多片段入门克隆。
对于LR反应，所有入门克隆都可以使用试剂盒内提供的对照入门克隆所替代。预期的克隆数和/或产生的克隆的表型可以被用来确定LR重组反应的效率。在极端情况下，如果目的载体attR位点不正确，那么LR反应将不会产生任何克隆。通过顺次进行载体替换反应，有问题的入门克隆可以被找出来。
•对于2片段LR Clonase II Plus阳性对照反应，如果使用全部10 µL LR反应进行转化，预期的克隆数为2,000 – 15,000
•对于3片段LR Clonase II Plus阳性对照反应，如果使用全部10 µL LR反应进行转化，预期的克隆数为1,000 – 5,000
•对于4片段LR Clonase II Plus阳性对照反应，如果使用全部10 µL LR反应进行转化，预期的克隆数为50 – 500
提供，我们提供pcDNA6.2/V5-PL-DEST载体，它是没有启动子的。如果您想使用MultiSite Gateway Pro试剂盒克隆您自己的5’启动子，那么您可以使用该载体。
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.
pDONR 221 is provided as a positive control for the BP recombination reaction, but cannot be used to generate multi-fragment entry clones in the MultiSite Gateway Pro kit.
For the LR reaction, all Entry clones can be substituted with the supplied Control Entry clones. The number of colonies expected and/or the phenotype of the resulting clones are used to determine the efficiency of the LR recombination reaction. In the unlikely event that the attR sites in the destination vector are incorrect, then the LR reaction will result in zero clones. By performing one-by-one vector substitution reactions, the entry clone that is flawed can be identified.
– For the 2-Fragment LR Clonase® II Plus Positive Control recombination reaction, 2,000 - 15,000 colonies are expected if the entire 10 µL LR reaction is transformed.
– For the 3-Fragment LR Clonase® II Plus Positive Control recombination reaction, 1,000 - 5,000 colonies are expected if the entire 10 µL LR reaction is transformed.
– For the 4-Fragment LR Clonase® II Plus Positive Control recombination reaction, 50 - 500 colonies are expected if the entire 10 µL LR reaction is transformed.
Yes, we carry pcDNA6.2/V5-PL-DEST, that is promoter-less. You would use this vector if you wanted to clone in your own 5' promoter using the MultiSite Gateway Pro kit.
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.