Product FAQ

Do I have to use your BLOCK-iT TOPO Transcription Kit to generate the long dsRNA as a template to work with BLOCK-iT Dicer?

Answer

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.

Answer Id: E4419

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Product FAQ

Is it possible to generate a full-length cDNA from mRNA attached to Dynabeads magnetic beads, and what reverse transcription kit do you recommend?

Answer

It is possible to generate full-length cDNA from mRNA attached to Dynabeads magnetic beads. We recommend a thermostable reverse transcription kit, so that difficult regions with GC-rich secondary structures are accommodated. However, it is not possible to start the reaction by heating the mRNA on the beads because that will elute the mRNA (A:T base pairs are the least thermostable).

We have used ThermoScript reverse transcriptase, inhouse, with Oligo(dT)25 on the beads as primers. The cDNA synthesis was performed according to the manufacturer's instructions. When using a thermostable reverse transcriptase and the Oligo(dT)25 as primer for first-strand cDNA synthesis, an initial step of incubation at 50 degrees C for 5 min is necessary before proceeding at the recommended elevated temperature. This is to start the cDNA synthesis beyond the A:T hybridization point so that the mRNA doesn't fall off the beads. The resulting cDNA is covalently attached to the bead surface, and the beads with the attached cDNA can be used as template in multiple hybridization reactions.

Answer Id: E6078

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Manual / Product Insert

User Guide: TranscriptAid T7 High Yield Transcription Kit

Version: Jan. 2015
Catalog #

Product FAQ

Can cDNA be generated using the High Capacity cDNA Reverse Transcription kit (SKU 4322171) for quantitative qPCR applications?

Answer

Yes, the single-stranded cDNA generated via the High-Capacity cDNA Reverse Transcription can be used in real-time PCR applications. In addition, we also recommend Superscript VILO cDNA Synthesis Kit, High Capacity RNA to cDNA kit, and/or High Capacity RNA to cDNA Master Mix for total RNA.

Answer Id: E2149

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Limited Use Label License

BLOCK-iT™ RNAi TOPO® Transcription Kit

Limited Use Label License
172
236
Catalog #
  • K350001(Discontinued)

Product FAQ

Can I order the random primers from the High-Capacity cDNA Reverse Transcription Kit separately?

Answer

No. The 10X random primers are only available as part of the High-Capacity Reverse Transcription Kit.

Answer Id: E7478

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Vector Data

BLOCK-iT T7-TOPO Linker

Vector Data

pcDNA1.2/V5-GW/lacZ

Product FAQ

Does the Lipofectamine MessengerMAX Transfection Reagent kit include reagents for in vitro transcription of mRNA?

Answer

Although it does not, we recommend the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Cat. No. AMB1345) to generate capped and tailed mRNA for highest mRNA stability.

Answer Id: E9029

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Product FAQ

When transfecting mRNA, does the packaging of the foreign RNA into vesicles reduce the accessibility of the mRNA for protein translation or is the mRNA all released in the cytoplasm? If packaged away, what percent is packaged vs. what percent is left to be available for translation? Is this observed with DNA plasmids?

Answer

We have not observed differences between how a cell packages an mRNA payload versus a DNA payload for the purpose of delivery. Transfection involves complex formation between a liposome and mRNA, which create lipoplexes that are taken up by the cell via endocytosis. The liposome protects the mRNA during this process and also assists in endosomal escape, which releases the mRNA into the cytoplasm of the cell. The mRNA is immediately available for translation with the ribosome. In vitro transcribed mRNA may be prepared using the mMESSAGE mMACHINE T7 Ultra Transcription Kit, which incorporates a 5’ ARCA cap and a 3’ poly(A) tail to mimic endogenously transcribed mRNA.

Answer Id: E9037

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Product FAQ

What is mechanism of the TaqMan MicroRNA Assays?

Answer

There are 2 steps involved in using the TaqMan MicroRNA Assays:

1. RT step: total RNA to cDNA using miRNA-specific RT primers from the TaqMan MicroRNA Assays and the TaqMan MicroRNA Reverse Transcription Kit (P/N 4366596, 200 reactions or 4366597, 1000 reactions)

2. PCR step: cDNA to PCR products using primers and probe from the TaqMan MicroRNA Assays together with TaqMan Universal PCR Master Mix No AmpErase UNG (P/N 4324018).

Answer Id: E2516

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Product FAQ

What can I do to improve the sensitivity of my qPCR assay?

Answer

If you are targeting a low-abundance gene, you may have trouble getting Ct values in a good, reliable range (Ct > 32). To increase the sensitivity of the assay, you may want to consider the following:

- Increase the amount of RNA input into your reverse transcription reaction, if possible
- Increase the amount of cDNA in your qPCR reaction (20% by volume max)
- Try a different reverse transcription kit, such as our SuperScript VILO Master Mix, for the highest cDNA yield possible
- Consider trying a one-step or Cells-to-CT type workflow (depending on your sample type)

Answer Id: E7510

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Product FAQ

How are the BLOCK-iT Dicer RNAi Transfection and BLOCK-iT Complete Dicer RNAi Kit configured?

Answer

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.

Answer Id: E4420

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Product FAQ

How easy is it to use Lipofectamine MessengerMAX Transfection Reagent?

Answer

Lipofectamine MessengerMAX Transfection Reagent has a very simple one-tube protocol (http://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_MessengerMAX_man.pdf). The mRNA can either be transcribed using the mMESSAGE mMACHINE ULTRA T7 Transcription Kit or can be purchased ready-to-use (e.g., GeneArt CRISPR Nuclease mRNA, Cat. No. A25640).

Answer Id: E9031

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