SAN TA C RUZ BI OTEC HNOL OG Y, INC . Dyskerin shRNA Plasmid (h): sc-38254-SH BACKGROUND STORAGE AND RESUSPENSION Dyskerin (NAP57) associates with the chaperone protein Nopp140 and forms Store lyophilized shRNA plasmid DNA at 4° C with desiccant. Stable for a small ribonucleoprotein particle with GAR1 (NOLA1), NHP2 (NOLA2) and at least one year from the date of shipment. Once resuspended, store at Nop10 for the isomerization of uridine to pseudouridine. GAR1, NHP2 and 4° C for short term storage or -80° C for long term storage. Avoid repeated Dyskerin localize to the dense fibrillar component of the nucleolus and in freeze thaw cycles. nuclear Cajal bodies. The Dyskerin gene maps to chromosome Xq28. Missense Resuspend lyophilized shRNA plasmid DNA in 200 µl of the deionized water mutations in the Dyskerin gene interfere with normal nuclear localization of provided. Resuspension of the shRNA plasmid DNA in 200 µl of deionized dyskerin and cause Dyskeratosis congenita (DKC). DKC is a rare, X-linked bone water makes a 0.1 µg/µl solution in a 10 mM Tris, 1 mM EDTA buffered marrow disorder characterized by cutaneous hyperpigmentation, dystrophy solu tion. of the nails, atrophy of the testicles and leukoplakia of the oral mucosa. The GAR1 gene maps to chromosome 4q25. The NHP2 gene maps to chromo - APPLICATIONS some 5q35.3 and encodes a 155-amino acid protein. Dyskerin shRNA Plasmid (h) is recommended for the inhibition of Dyskerin REFERENCES expression in human cells. 1. Hassock, S., et al. 1999. Mapping and characterization of the X-linked SUPPORT REAGENTS dyskeratosis congenita (DKC) gene. Genomics 55: 21-27. For optimal shRNA Plasmid transfection efficiency, Santa Cruz Biotechnology’s 2. Heiss, N.S., et al. 1999. Dyskerin localizes to the nucleolus and its mislo - shRNA Plasmid Transfection Reagent: sc-108061 (0.2 ml) and shRNA Plasmid calization is unlikely to play a role in the pathogenesis of dyskeratosis Transfection Medium: sc-108062 (20 ml) are recommended. Control shRNAs congenita. Hum. Mol. Genet. 8: 2515-2524. are available as 20 µg lyophilized plasmid DNA. Each encodes a scrambled 3. Dragon, F., et al. 2000. In vitro assembly of human H/ACA small nucleolar shRNA sequence that will not lead to the specific degradation of any known RNPs reveals unique features of U17 and telomerase RNAs. Mol. Cell. Biol. cel lular mRNA. Control shRNA Plasmids include: sc-108060, sc-108065 and 20: 3037-3048. sc- 108066. 4. Pogacic, V., et al. 2000. Human H/ACA small nucleolar RNPs and telom erase GENE EXPRESSION MONITORING share evolutionarily conserved proteins NHP2 and NOP10. Mol. Cell. Biol. 20: 9028-9040. Dyskerin (H-3): sc-373956 is recommended as a control antibody for moni - toring of Dyskerin gene expression knockdown by Western Blotting (start ing 5. Wang, C., et al. 2002. Immunopurified small nucleolar ribonucleoprotein dilution 1:200, dilution range 1:100-1:1000) or immunofluorescence (starting particles pseudouridylate rRNA independently of their association with dilution 1:50, dilution range 1:50-1:500). phosphorylated Nopp140. Mol. Cell. Biol. 22: 8457-8466. To ensure optimal results, the following support reagents are recommended: CHROMOSOMAL LOCATION 1) Western Blotting: use m-IgG κ BP-HRP: sc-516102 or m-IgG κ BP-HRP (Cruz Marker): sc-516102-CM (dilution range: 1:1000-1:10000), Cruz Marker™ Genetic locus: DKC1 (human) mapping to Xq28. Molecular Weight Standards: sc-2035, UltraCruz ® Blocking Reagent: sc-516214 and Western Blotting Luminol Reagent: sc-2048. 2) Immunofluo- PRODUCT rescence: use m-IgG κ BP-FITC: sc-516140 or m-IgG κ BP-PE: sc-516141 Dyskerin shRNA Plasmid (h) is a pool of 3 target-specific lentiviral vector (dilution range: 1:50-1:200) with UltraCruz ® Mounting Medium: sc-24941 plasmids each encoding 19-25 nt (plus hairpin) shRNAs designed to knock or UltraCruz ® Hard-set Mounting Medium: sc-359850. down gene expression. Each plasmid contains a puromycin resistance gene for the selection of cells stably expressing shRNA. Each vial contains 20 µg RT-PCR REAGENTS of lyophilized shRNA plasmid DNA. Suitable for up to 20 transfections. Also Semi-quantitative RT-PCR may be performed to monitor Dyskerin gene see Dyskerin siRNA (h): sc-38254 and Dyskerin shRNA (h) Lentiviral Particles: expression knockdown using RT-PCR Primer: Dyskerin (h)-PR: sc-38254-PR sc-38254-V as alternate gene silencing products. (20 µl, 422 bp). Annealing temperature for the primers should be 55-60° C and the extension temperature should be 68-72° C. RESEARCH USE The purchase of this product conveys to the buyer the nontransferable right PROTOCOLS to use the purchased amount of the product and all replicates and derivatives See our web site at www.scbt.com for detailed protocols and support for research purposes conducted by the buyer in his laboratory only (whether products. the buyer is an academic or for-profit entity). The buyer cannot sell or other - wise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party, or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com.