The touchscreen shows the voltage measured on the chip and there will be some contrast according the signal intensity which may look like bubbles.

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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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Perform a kill curve to determine the antibiotic sensitivity of your cell line. Ensure that viral stocks are stored properly at -80 degrees C, and do not undergo freeze/thaw more than 3 times. Lastly, transducer the lentiviral contruct into cells in the presence of Polybrene reagent.

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Our siRNA libraries are compatible with most automated liquid handling robots including Tecan, Hamilton, and custom-modified robots. Our R&D teams use Tecan liquid handlers with the following plates:

- 96-well plates, Axygen Cat. No. PCR-96-FS-C, 96 Well Full Skirt PCR Microplates
- 384-well plates, Axygen Cat. No. P-384-120SQ-C, 120 µL 384 Deep Well "Diamond Plate" Microplate with Square Wells, Clear.

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Once you have assembled all the necessary plasticware and diluents, the siRNA can be distributed into sterile tissue culture plates suitable for RNAi screening protocols. We recommend that you aliquot the siRNA as a droplet onto the working surface of the sterile tissue culture plate, then seal and freeze the plates at -20 degrees C immediately to reduce risk of contamination or excessive evaporation. siRNA that evaporate are more difficult to complex with most transfection reagents which can lead to decreased transfection efficiencies. You can aliquot larger volume with less concentrated working stock if excessive evaporation is observed in your operation. In most cases, up to 20 µL of siRNA for a 96-well plate, or 10 µL of siRNA for a 384-well plate, is still compatible with a wide variety of cells and transfection reagents.

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RNA oligonucleotides are susceptible to degradation by exogenous ribonucleases introduced during handling. Wear gloves when handling this product. Use RNase-free reagents, tubes, and barrier pipette tips. Upon receipt, your siRNAs may be safely stored in a non-frost-free freezer at or below -20 degrees C (dried oligonucleotides are shipped at ambient temperature). Invitrogen siRNA reagents are shipped as dry pellets at ambient temperature and should be stored at -20 degrees C upon arrival in a manual defrost or noncycling freezer. Under these conditions, the siRNAs are stable for at least 3 years. If necessary siRNAs as dry pellets (unopened) can be stored at 4 degrees C for at least a year.

To resuspend Invitrogen siRNAs provided in plates:
Centrifuge each plate at low speed (maximum RCF 4,000 x g) to collect the contents at the bottom of the wells before removing the seal.
Wipe the adhesive foil cover with 70% ethanol or other RNase-decontamination solution such as RNaseZap RNase Decontamination Solution (Cat. No. AM9780, AM9782, AM9784).
1. Thermo Scientific or carefully peel back the foil seal to gain access to wells. Use caution and avoid shredding the seal.
2. Add nuclease-free, sterile water, using a multichannel pipettor or liquid handling system and sterile tips, to achieve the desired concentration. Resuspend siRNAs to a convenient stock concentration using the recommended volume of Invitrogen Nuclease-Free Water (not DEPC-treated). Concentrated stocks of 10 µM or more are recommended. However, stock solutions of 2-5 µM may better accommodate dilution schemes for high?throughput transfections and assays conducted on robotic platforms.
3. Gently pipet up and down 5 times to resuspend. Place the solution on an orbital mixer/shaker for 70-90 minutes at room temperature. This additional mixing results in more reliable resuspension.
4. Centrifuge briefly to collect the liquid at the bottom of the wells, if necessary.
5. (Optional) Aliquot the siRNAs into one or more daughter plates, to limit the number of freeze/thaw cycles to which the siRNAs are subjected.
6. Store at -20 degrees C or lower for long-term storage. The siRNAs can be stored at 4 degrees C for short-term use, but care should be taken to seal well to avoid evaporation.

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siRNAs in the Silencer Select Human Genome siRNA Library V4 correspond to each of 21,584 genes, with 3 unique, nonoverlapping siRNAs provided per target, for a total of 64,752 siRNAs. The so called target genes correspond to greater than 98% of genes listed by NCBI that have at least one or more curated RefSeq coding transcripts. The siRNAs were designed to hit all RefSeq coding transcripts of that gene that were known at the time of design. The siRNAs targeting the druggable portion of this library are arranged by gene functional class to enable easy screening of important gene subsets.

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The Silencer Select Human Genome siRNA Library and its subsets (Silencer Select Human Druggable Genome, Silencer Select Human Druggable Genome Extension Set, and Silencer Select Human Genome Extension Set) are plated in 384-well plates, each at 0.25 nmol siRNA per well. Each plate has the last 2 columns empty, and siRNAs to the same target are plated in different plates in the same well location. All other premade Invitrogen siRNA libraries are plated at 0.25 nmol siRNA per well in 96-well plates. Each plate has the last column empty, and siRNAs to the same target are plated in different plates in the same well location.

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Below are some citations that reference Invitrogen siRNA libraries/collections. This is only a subset. Please contact us for an updated citation list at rnailibraries@lifetech.com.

Silencer Select siRNA
Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of Src kinases. Ritzerfeld J, Remmele S, Wang T et al. (2011) Genome Res Jul 27 (available online)
Identification of protein kinases that control ovarian hormone release by selective siRNAs. Sirotkin AV, Ovcharenko D, Mlyncek M (2010) J Mol Endocrinol 44:45-53.
Selection of hyperfunctional siRNAs with improved potency and specificity. Wang X, Xiaohui Wang X, Varma RK et al. (2009) Nucleic Acids Res:37: e152.
Protein kinases controlling PCNA and p53 expression in human ovarian cells. Alexander V Sirotkin AV, Ovcharenko D, Benco A et al. (2009) Funct Integr Genomics 9:185-195.

Silencer siRNA
Identification of host factors involved in borna disease virus cell entry through a small interfering RNA functional genetic screen. Clemente R, Sisman E, Aza-Blanc P et al. (2010) J Virol 84:3562-3575.
siRNA screening reveals JNK2 as an evolutionary conserved regulator of triglyceride homeostasis. Grimard V, Massier J, Richter D et al. (2008) J Lipid Res 49:2427-2440.
Polo-like kinase 2 (PLK2) phosphorylates alpha-synuclein at serine 129 in central nervous system. (2009) Inglis KJ, Chereau D, Brigham EF et al. J Biol Chem 284:2598-2602.
Identification of survival genes in human glioblastoma cells by small interfering RNA screening. Thaker NG, Zhang F, McDonald PR et al. (2009) Mol Pharmacol 76:1246-1255.
Low-dose arsenic trioxide sensitizes glucocorticoid-resistant acute lymphoblastic leukemia cells to dexamethasone via an Akt-dependent pathway. Bornhauser BC, Bonapace L, Lindholm D et al. (2007) Blood 110:2084-2091.
Visual screening and analysis for kinase-regulated membrane trafficking pathways that are involved in extensive beta-amyloid secretion. Adachi A, Kano F, Saido TC et al. (2009) Genes Cells 14:355-369.
Identification and characterization of 3-iodothyronamine intracellular transport. Ianculescu AG, Giacomini KM, Scanlan TS (2009) Endocrinology 150:1991-1999. High-throughput RNAi screening in vitro: from cell lines to primary cells. Ovcharenko D, Jarvis R, Hunicke-Smith S et al. (2005) RNA 11:985-993.

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If you still would like to have 4 siRNAs in your library instead of 3, you have the option of adding 1 siRNA from our Silencer designs for the human library.

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The TaqMan GMO Screening kit is designed to perform four reactions for each sample analysed (P35S, TNOS, P34S-FMV, and plant).

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