DIG Northern Starter Kit.pdf

DIG Northern Starter Kit y Version 8.0 Content version: August 2010 For transcription-labeling of RNA with digoxigenein and SP6/T7/T3 RNA polymerases and chemiluminescent detection with CDP-Star, ready-to-use. Cat. No. 12 039 672 910 Kit for 10 labeling reactions and detection of 10 blots of 10  10 cm2 Store the kit at 15 to 25°C For life science research only. Not for use in diagnostics procedures. FOR IN VITRO USE ONLY. 2 12 039 672 910 DIG Northern Starter Kit y Version 8.0 1. Preface 1.1 Table of Contents 1. Preface 2 1.1 Table of Contents 2 1.2 Kit contents 3 2. Introduction 5 2.1 Product overview 5 3. Procedures and required materials 8 3.1 Before you begin 8 3.2 Flow chart 8 3.3 DNA template preparation 9 3.4 DIG-RNA labeling 11 3.5 Determination of labeling efficiency 12 3.6 Gel system suitable for DIG Northern blots 15 3.7 RNA transfer and fixation 16 3.8 Hybridization 17 3.9 Immunological detection 19 3.10 Stripping and reprobing of RNA blots 21 4. Results 22 4.1 Typical results 22 5. Appendix 24 5.1 Trouble shooting 24 5.2 Changes to previous version 25 5.3 References 26 5.4 Ordering Information 26 3 12 039 672 910 DIG Northern Starter Kit y Version 8.0 1.2 Kit contents Bottle/ Cap Label Content including function 1a Labeling Mix ? 40 l ? 5 × conc. labeling mixture containing optimal concentrations of unlabeled nucleotides and DIG -11-UTP ? Clear viscous solution ? For efficient in vitro transcription of linearized template DNA 1b Transcription Buffer ? 40 l ? 5 × conc. transcription buffer ? Clear solution ? For efficient in vitro transcription of linearized template DNA 2 SP6 RNA Polymerase ? 20 l ? [20 U/l] ? Synthezise RNA from a DNA template 3 T7 RNA Polymerase ? 20 l ? [20 U/l] ? Synthezise RNA from a DNA template 4 T3 RNA Polymerase ? 20 l ? [20 U/l] ? Synthezise RNA from a DNA template 5 Anti-digoxigenin-AP, Fab fragments ? 60 l ? [750 U/ml] ? Polyclonal sheep anti-digoxigenin, Fab-fragments, conjugated to