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Perform a DraI digestion and self ligation of the vector to form a pcDNA6.2-GW/miR clone expressing the same pre-miRNA. See page 40 of the manual for a more detailed protocol.

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Use of the BLOCK-iT Lentiviral RNAi Expression System to facilitate lentiviral based delivery of shRNA to mammalian cells provides the following advantages:

- The pENTR/U6 entry vector provides a rapid and efficient way to clone ds oligo duplexes encoding a desired shRNA target sequence into a vector containing an RNA Pol III-dependent expression cassette (i.e., U6 RNAi cassette) for use in RNAi analysis.
- The vectors in the System are Gateway-adapted for easy recombination of the U6 RNAi cassette from the pENTR/U6 vector into the pLenti6/BLOCK-iT-DEST vector.
- Generates a replication-incompetent lentivirus that effectively transduces both dividing and nondividing mammalian cells, thus broadening the potential RNAi applications beyond those of other traditional retroviral systems (Naldini, 1998).
- Efficiently delivers the shRNA of interest to mammalian cells in culture or in vivo.
- Provides stable, long-term expression of the shRNA of interest beyond that offered by traditional adenovirus-based systems.
- Produces a pseudotyped virus with a broadened host range (Yee, 1999).
- Includes multiple features designed to enhance the biosafety of the system.

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Yes, we offer 4 T-REx cell lines: 293, HeLa, CHO, or Jurkat cells. Alternatively, you can create your own T-REx cell line that stably expresses the Tet repressor form the pcDNA6/TR.

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Please review the possibilities below:

- Single-stranded oligos designed incorrectly; verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top strand oligo.
- Ensure that oligos are annealed at room temp for 5-10 minutes after heating to 95 degrees C.
- Check the molar ratio you are using for annealing top and bottom-strand oligo; equal amounts should be used.

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Yes, as long as you do not use a cell line expressing the Tet repressor, expressing will be constitutive.

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Please see the steps below:

Clone the double-stranded DNA oligo encoding an shRNA or miR RNAi into one of the BLOCK-iT entry (shRNA) or expression (miR RNAi) vectors.
Transfer the RNAi cassette into the adenoviral (shRNA only) or lentiviral destination vector by Gateway recombination.
Transfect RNAi vectors into the viral producer cells to produce viral stocks, which can be used immediately or stored at -80 degrees C.
Harvest viral supernatants and determine the titer (amplify adenoviral stocks if desired).
Transduce lentiviral or adenoviral stocks to any cell type.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5' end, while the bottom-strand oligo should include AAAA on the 5' end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5' end and that the bottom-strand oligo includes CCTG at the 5' end.

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Low expression levels can be due to several factors. Please see the suggestions below:

- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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The target sequence used may contain strong homology to other genes; please select a different target region.

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Identity: The mass of a sample of each single-stranded RNA oligonucleotide is analyzed using MALDI-TOF mass spectrometry and compared to the calculated mass.
Annealing: A sample of the annealed siRNA is analyzed by nondenaturing gel electrophoresis.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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Invitrogen siRNA libraries are shipped dried and are therefore shipped at ambient temperature. Although dried siRNAs are remarkably stable, we recommend that you store the dried siRNAs at -20 degrees C (or lower) until ready for use.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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Efficient and reproducible transfection is critical for any siRNA experiment. Success or failure of a siRNA experiment often hinges on siRNA delivery. Optimizing siRNA delivery and cell viability during transfection eliminates the most common causes of unsuccessful gene silencing experiments. Optimal transfection conditions vary depending on cell line. As a result, it is imperative to optimize transfection conditions for your cells before screening a siRNA library to empirically determine the highest levels of gene silencing while minimizing toxicity associated with the transfection process.

Investing the time up front to identify the best transfection conditions for your experimental system will limit transfection variability and maximize data quality by maximizing siRNA uptake and minimizing cell toxicity. Once you have identified an optimized transfection protocol, the interesting and productive work of library screening can begin.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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The Silencer Select Human Genome Library can be purchase in 384-well plates or 96-well plates. There are 186 384-well plates or 738 96-well plates in the respective library formats. Each 384-well plate has the last 2 columns empty, while the 96-well plate has the last column empty.

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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Contact your local sales representative or contact us at rnailibraries@lifetech.com. We will provide you with a Quote Request form to indicate exactly how you would like your siRNA library formatted. You will need to be prepared to provide the following information:

Your gene/target list (preferably by NCBI Entrez Gene ID; RefSeq mRNA Accession Number is also acceptable)
Your choice of Silencer Select siRNA, Silencer siRNA (unmodified), or Invitrogen In Vivo siRNA
The number of siRNAs needed per target (typically 3 for coding genes)
The amount of siRNA per well desired:
Silencer Select siRNAs available as 0.1, 0.25, 1, 2, or 5 nmol
Silencer siRNAs available as 1, 2, or 5 nmol
Your desired plating format

Find additional tips, troubleshooting help, and resources within our RNAi Support Center.

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The Low Input RiboMinus Eukaryote kit improves upon the previous system (RiboMinus Eukaryote Kit for RNA-Seq) by removing significantly more rRNA (both cytoplasmic and mitochondrial) from the total RNA. This system provides maximum mapping to reference database with minimum bias, maintaining relative gene expression levels of even smaller RNA species. The resulting total RNA after depletion contains minimum contaminating rRNA with reduced variability.

The Low Input RiboMinus Eukaryote System v2 is designed for the removal of human, mouse, and rat rRNA. A magnetic bead-based purification module is included for purification of the whole transcriptome without altering the expression levels of small or non-coding RNA.

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