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View additional product information for MultiSite Gateway® Pro 2.0, for cloning two DNA fragments into a Gateway® destination vector - FAQs (12537102)
13 product FAQs found
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.