Jan 14, 2020
- 1Imperial College London
Protocol Citation: Ida Barlow 2020. In vitro transcription of DIG-labelled RNA probe. protocols.io https://dx.doi.org/10.17504/protocols.io.ba4pigvn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 08, 2020
Last Modified: January 14, 2020
Protocol Integer ID: 31599
Abstract
Protocol for making DIG-labelled RNA probes suitable for in situ hybridisation
Materials
MATERIALS
DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #D0632
T3 RNA Polymerase, 1,000uPromegaCatalog #P2083
DNaseINEB
RNA clean & concentrator-25Zymo ResearchCatalog #R1017
RNase InhibitorThermo FisherCatalog #N8080119
Digoxigenin-11-UTPMerck Millipore (EMD Millipore)Catalog #11209256910
Assemble reaction on ice:
2 µL DTT
2 µL 10X DIG-NTP mix (5 mM)
0.5 µL RNAse inhibitor
2 µL 10 X transcription buffer
1 µg linearlised template
1 µL RNA polymerase
make up to 20ul total volume with WATER
20 µL TOTAL volume
Note
5mM DIG-NTP mix (10X):
2 µL 100mM CTP
2 µL 100mM GTP
2 µL 100mM ATP
1.3 µL 100mM UTP
7 µL 10mM DIG-11-UTP
25.7 µL Nuclease-free water
Mix well by gently flicking and spin down tube contents
Incubate at 37 °C for 02:00:00
Add 1 µL DNaseI and digest the template at 37 °C for 00:15:00
Use ZYMO RNA cleanup kit to purify RNA and check quality by agarose gel electrophoresis; quantiy with nanodrop spectrophotometer