For general laboratory use. FOR IN VITRO USE ONLY. Biotin RNA Labeling Mix, 10 × conc. RNA labeling with biotin-16-UTP by in vitro transcription with SP6, T7 and T3 RNA polymerases for 20 transcriptions Cat. No. 11 685 597 910 40 ␮l Version Nov. 2004 Store at ⫺15 to ⫺25° C 1. Product overview Contents Sufficient for 20 transcriptions. Label Biotin RNA Labeling Mix, 10 × conc. 2. Procedures and required materials Content • 40 ␮l • 10 × conc. NTP labeling mixture: 10 mM ATP, 10 mM CTP, 10 mM GTP, 6.5 mM UTP, 3.5 mM Biotin-16-UTP, pH 7.5 (20°C) Labeling principle Biotin-16-UTP is incorporated by SP6, T7 and T3 RNA polymerases at approx. every 20-25th nucleotide of the transcript under the conditions described below (1). The Biotin RNA Labeling Mix is especially designed for the use with Roche Biochemicals SP6, T7 and T3 RNA polymerases, which are supplied with an optimized transcription buffer. Sample material Application • linearized plasmid DNA The DNA to be transcribed is cloned into the polylinker site of an appropriate transcription vector which contains adjacent to the polylinker a promoter for SP6, T7 or T3 RNA polymerase (2,3). For the synthesis of 'run off' transcripts the plasmid is linearized by a restriction enzyme. Restriction enzymes creating 5'-overhangs should be used; 3’ overhangs should be avoided. The linearized template DNA should be purified by phenolchloroform extraction and ethanol precipitation, to avoid RNase contamination. For 'run around' transcription circular plasmid DNA is used. • PCR product PCR-fragments which contain RNA polymerase promoter sequences can also act as templates for transcription. Purification of the correct PCR fragment by gel electrophoresis prior to transcription is recommended. Biotin-labeled RNA is used for hybridization to • Northern blots • Southern blots • plaque or colony lifts or • RNase protection experiments • in situ hybridization Labeling efficiency In the standard reaction about 10 ␮g ‘full length’ biotin labeled RNA is synthesized from 1 ␮g linearized plasmid DNA with an insert of approx. 1 kb. Larger amounts of biotin-labeled RNA can be obtained by scaling up the reaction components. The amount of synthesized labeled RNA depends on the amount, size (site of linearization) and purity of the template DNA. Note: Longer incubations do not increase the yield of labeled RNA. Stability The unopened vial is stable at ⫺15 to ⫺25° C through the expiration date printed on the label. Note: Repeated freezing and thawing should be avoided. To avoid contamination we recommend to aliquot the Biotin RNA labeling Mix solution and to store in 2-3 portions. 1104.11693 069 ➅ 2.1 Standard labeling assay Additional • SP6 RNA polymerase* or equipment and • T7 RNA polymerase* or reagents required • T3 RNA polymerase* • DNase, RNase free* (optional) • Transcription buffer 10 × conc., is supplied with the RNA polymerases: 400 mM Tris-HCl, pH 8.0 (20° C); 60 mM MgCl, 100 mM dithiotreitol (DTT), 20 mM spermidin. Procedure Standard labeling assay. Note: Please make sure, you work under RNase-free conditions. Step 1 Action • Add the following to a microfuge tube on ice: Reagent Volume 1 ␮g linearized plasmid DNA or appropriate amount of PCR product (100-200 ng) x ␮l Biotin RNA labeling mix, 10 × 2 ␮l 10 × transcription buffer 2 ␮l add sterile RNase free double dist. water to a final volume of 18 ␮l x ␮l RNA polymerase (SP6, T7 or T3) 2 ␮l • Mix and centrifuge briefly. • Incubate for 2 h at 37°C. 2 • Add 2 ␮l DNase I, RNase-free to remove template DNA. (optional) • Incubate for 15 min at 37° C. Note: Only required for RNase-protection experiments! 3 Stop the reaction by adding 2 ␮l 0.2 M EDTA (pH 8.0). 4 Use the labeled probe immediately or store ethanol precipitated at ⫺15 to ⫺25° C or ⫺60° C or below. DNase treatment When the biotin-labeled RNA is used for hybridization to Northern or Southern blots, plaque or colony lifts a DNase-treatment is not required, as the amount of biotin-labeled RNA transcript is far in excess of the template DNA. 2.2 General remarks on usage and application Analysis of labeled RNA Quality and quantity of the transcript can be analyzed by non-denaturing agarose gel electrophoresis and ethidium bromide staining. The signal from the RNA band should be stronger than that from the DNA. The size and the amount of the transcript can be estimated by comparison to known RNAs. Hybridization with Add 0.2-1 ␮l (approx. 20 -100 ng) of the biotin-labeled labeled RNA RNA per ml hybridization solution. Detection of biotin-labeled RNA probes After hybridization to nucleic acid targets bound to nylon membrane, the biotin label is detected by an immunoassay with anti-biotin-AP in the Biotin Luminescent Detection Kit*, or Streptavidin-AP conjugate and the color substrates NBT/BCIP* or the chemiluminescent substrates CSPD1) or CDP-Star2). For in situ hybridizations, avidin, conjugated to fluorophores is used. Single reagents Product Pack Size Anti-biotin-AP, Fab fragments 11 426 303 001 Avidin-Fluorescein 1 mg 11 975 595 910 Avidin-Rhodamine 1 mg 11 975 609 910 CDP-Star, ready-to-use 2x 50 ml 12 041 677 001 CSPD, ready-to use 2x 50 ml 11 755 633 001 DIG RNA Labeling Mix 40 ␮l 11 277 073 910 DIG-labeled control RNA 50 ␮l 11 585 746 910 10 000 units 10 776 785 001 Hybridization bags 50 bags 11 666 649 001 Klenow Enzyme 100 units 500 units 11 008 404 001 11 008 412 001 DNase I, RNase free NBT/BCIP Stock Solution Nylon Membrane, positively charged 3. Appendix 3.1 References 1 Höltke, H.J. & Kessler, C. (1990) Nucleic Acid Res. 18, 5843. 2 Dunn, J. J. & Studier, F. W. (1983) J. Mol. Biol. 166, 477. 3 Kasavetis, G. A. et al. (1982) J. Biol. Chem. 257, 5779 3.2 Ordering Information Protector RNase Inhibitor DIG Wash and Block Buffer Set Biotin Luminescent Detection Kit Pack Size Cat. No 1 kit 11 175 025 910 (2 × 10 labeling reactions) 30 blots 11 585 762 001 (10 × 10 cm2) 1 kit (50 blots) 11 811 592 910 8 ml 11 681 451 001 10 sheets (20 × 30 cm) 20 sheets (10 × 15 cm) 1 roll (0.3 × 3 m roll) 11 209 272 001 10 000 units 2 000 units 03 335 402 001 03 335 399 001 11 209 299 001 11 417 240 001 SP6 RNA polymerase 1000 units 10 810 274 001 T3 RNA polymerase 1000 units 5000 units 11 031 163 001 11 031 171 001 T7 RNA polymerase 1000 units 5000 units 10 881 767 001 10 881 775 001 Kits Product DIG RNA Labeling Kit (SP6/T7) Cat. No. 150 units (200 ␮l) *available from Roche Applied Science. 1) CSPD is a trademark of Tropix, Inc. Bedford, MA, USA and covered by European patent application 0497 972 and U.S. patent 5112960, both assigned to Tropix Inc. 2) CDP-Star is a trademark of Tropix, Inc., Bedford, MA, USA and covered under U.S. patent 5,326,882. DIG-11-UTP is covered by the following patents, EP patent 0324474 and US 5,344,757 granted to Roche Applied Science How to contact Roche Applied Science www.roche-applied-science.com to order, solve technical queries, find product information, or contact your local sales representative. www.roche-applied-science.com/pack-insert/11685597910a.pdf Please visit our new Online Technical Support Site under www.roche-applied-science.com/support Roche Diagnostics GmbH Roche Applied Science Nonnenwald 2 82372 Penzberg Germany