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  • Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture.

Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture.

Bio-protocol (2018-03-05)
Imre Gaspar, Frank Wippich, Anne Ephrussi
ABSTRACT

Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
(+)-Biotin N-hydroxysuccinimide ester, ≥98% (HPLC), powder
Sigma-Aldrich
Trizma® base, Primary Standard and Buffer, ≥99.9% (titration), crystalline
Sigma-Aldrich
Sodium acetate, anhydrous, for molecular biology, ≥99%
Sigma-Aldrich
N,N,N′,N′-Tetramethylethylenediamine, BioReagent, suitable for electrophoresis, ≥99.0%
Sigma-Aldrich
Urea, powder, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Colorimetric Biotin Assay Kit, sufficient for 200 colorimetric assays