Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared
from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The
DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids.
DIG Labeling of cDNA Probes Protocol Protocol for DIG labeling of cDNA probes. Includes: Incorporation DIG during PCR; Estimating the yield of DIG labeled probes; DNA Dot Blotting; Prehybridization and Hybridization.
DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy.
Identification of Single Bacterial Cells using DIG-Labeled Oligonucleotides Protocol Protocol for the identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides; Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments; Detection of DIG-labeled oligonucleotide.
In Situ Hybridization to Human Metaphase Chromosomes using DIG-, Biotin- or Fluorochrome-Labeled DNA Protocol for In situ hybridization to human metaphase chromosomes using DIG-, biotin-, or fluorochrome-labeled DNA probes and detection with fluorochrome conjugates. Includes: Pretreatment of metaphase spreads on slides; Denaturation and hybridization; Single color fluorescent detection with immunological amplification; Multicolor fluorescence in situ hybridization (Multicolor FISH); Results obtained with human metaphase chromosome spreads.