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查看更多产品信息 BLOCK-iT™ RNAi TOPO® Transcription Kit - FAQs (K350001)
5 个常见问题解答
These kits are configured so that everything you need for your RNAi experiment is included. You just provide the dsRNA substrate or use the BLOCK-iTRNAi TOPO Transcription Kit (included in the BLOCK-iT Complete Dicer RNAi Kit) to generate your dsRNA. The BLOCK-iT Dicer RNAi Transfection kit provides the necessary reagents to generate enough diced product to do up to 150 transfection experiments in 24-well plate with up to five genes. This is significantly more transfection experiments than other Dicer kits.
The BLOCK-iT Dicer RNAi Transfection Kit includes:
(1) A high-quality preparation of BLOCK-iTDicer Enzyme RNAi
(2) Purification reagents to purify the diced siRNA (d-siRNA)
(3) Lipofectamine 2000 for high efficiency delivery of d-siRNA to mammalian cells
If you wish to use truly optimized Invitrogen reagents to prepare your dsRNA substrate and to complete the dicing reaction, we recommend the use of the BLOCK-iT Complete Dicer RNAi. An easy-to-use RNAi purification module with optimized protocols for isolating the dsRNA (BLOCK-iT RNAi TOPO Transcription Kit) or d-siRNA (BLOCK-iT Dicer RNAi Transfection Kit) is included with these kits. These are critical for best results. The purity of long dsRNA can affect how well this will act as a substrate for the dicing reaction. The optimized purification of the d-siRNA is essential, since if this is not purified completely away from the long dsRNA a general cell shutdown response can be triggered. Lipofectamine 2000 is also included in our BLOCK-iT Dicer RNAi Transfection Kit to assure high efficiency delivery and experimental success.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
While the BLOCK-iT TOPO Transcription kit is ideal for generating high yields of quality dsRNA for use as a Dicer substrate, long dsRNA from other sources will work as well.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
The RNAse III enzyme apparently cleaves long dsRNA into smaller (12-15 nucleotide) fragments than Dicer. There are some reports that these resulting fragments may be able to generate an RNAi effect, but in most cases much more of the product needs to be transfected to achieve such knockdown.
The Dicer enzyme is expected to be more effective, as it is a natural component of the endogenous RNAi pathway in eukaryotes, and purified recombinant Dicer used in an optimal protocol will cleave the long dsRNA to the specific size known to be most effective for generating the knockdown response.
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
Dicer can be a simple and inexpensive tool to use early in RNAi analysis. It is best suited for broad applications, such as:
- An initial screen of knockdown to several genes to select the ones of most interest to follow up on
- Knocking out all splice variants or all the family members of a particular gene
Find additional tips, troubleshooting help, and resources within our RNAi Support Center.
In addition to the CAAT and TATA boxes, the CMV promoter in pcDNA3.1 vector contains sequence homologous to base pairs 137 to 724 of the sequence submitted by Boshart, et al (GenBank Accession # K03104). The complete enhancer region is contained between 214 and 620 of this sequence. Therefore, by this definition, the CMV promoter could be said to be "complete".
Please note that this promoter does not contain an intron. Some people believe that the complete promoter must contain the intron, but that has not been demonstrated to be necessary for expression.