Preparation of randomly Digoxigenin-modified RNA probes by in vitro transcription with Dioxigenin-11-UTP
Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
RNT-101-DIGX | 30 reactions x 20 μl | 340,80 | Add to Basket/Quote Add to Notepad |
For general laboratory use.
Shipping: shipped on gel packs
Storage Conditions: store at -20 °C
avoid freeze/thaw cycles
Shelf Life: 12 months
Description:
HighYield T7 Digoxigenin RNA Labeling Kit (UTP-based) is designed to produce randomly Digoxigenin-modified RNA probes via in vitro transcription. Such probes are ideally suited for in situ hybridization and Northern Blot experiments. The labeling principle is similar to the underlying labeling principles of DIG RNA Labeling Mix (Roche) and DIG RNA Labeling Kit (Roche).
DIG-11-UTP is efficiently incorporated into RNA as substitute for its natural counterpart UTP using an optimized reaction buffer and T7 RNA Labeling Polymerase Mix.
35 % DIG-11-UTP substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of DIG-11-UTP/UTP ratio however, can easily be achieved with the single nucleotide format.
The resulting Digoxigenin-modified RNA probe can subsequently be detected by HRP- or AP-modified Digoxigenin antibody.
The kit contains sufficient reagents for 30 labeling reactions of 20 μl each (35 % DIG-11-UTP substitution, 1 mM ATP, GTP, CTP, 0.65 mM UTP, 0.35 mM DIG-11-UTP).
Content:
HighYield T7 RNA Labeling Polymerase Mix
2x 40 μl, incl. RNase inhibitor and 50 % glycerol (v/v)
HighYield T7 Reaction Buffer
1x 200 μl (10x), HEPES-based
ATP - Solution
1x 100 μl (100 mM)
GTP - Solution
1x 100 μl (100 mM)
CTP - Solution
1x 100 μl (100 mM)
UTP - Solution
1x 100 μl (100 mM)
DIG-11-UTP
1x 25 μl (10 mM)
T7 G-initiating control template (1.4 kbp)
1x 10 μl (200 ng/μl), 1.4 kbp PCR fragment plus T7 class III phi6.5 promotor resulting in approx. 1400 nt RNA transcript
PCR-grade water
1x 1.2 ml
DTT
1x 150 μl (100 mM)
To be provided by user
T7 Promotor-containing DNA template
RNA purification tools
RNAse-free DNAse I (optional)
1. Important Notes (Read before starting)
1.1 Prevention of RNAse contamination
Although a potent RNase Inhibitor is included, creating a RNAse-free work environment and maintaining RNAse-free solutions is critical for performing successful in vitro transcription reactions. We therefore recommend
1.2 Template requirements
T7 class III phi6.5 promotor
5'-TAATACGACTCACTATAGNN...-3’
Bold: First base incorporated into RNA, NN: ideally CG
or
T7 class II phi2.5 promotor
5'-TAATACGACTCACTATTAGNN...-3'
Bold: First base incorporated into RNA, NN: ideally CG
2. Preparation of working solutions
2.1 Preparation of 10 mM ATP/CTP/GTP working solution
2.2 Preparation of 10 mM UTP working solution
3. In vitro Transcription protocol
The protocol is optimized for 0.5 μg - 1 μg DNA template.
An optimal balance between reaction and labeling efficiency is typically achieved with 35% DIG-11-UTP substitution following the standard protocol below however, individual optimization might improve results for individual applications (e.g. 2.5 mM ATP, CTP, GTP, 0.2 mM UTP, 0.1 mM DIG-11-UTP)
Component | Volume | Final concenctration |
PCR-grade water | X μl | |
HighYield T7 Reaction Buffer (10x) | 2 μl | 1x |
100 mM DTT | 2 μl | |
10 mM ATP/CTP/ GTP working solution (s. 2.1) | 2 μl | 1 mM |
10 mM UTP working solution (s. 2.2) | 1.3 μl | 0.65 mM |
10 mM DIG-11-UTP | 0.7 μl | 0.35 mM |
Template DNA | X μl | 0.5 - 1 μg |
HighYield T7 RNA Labeling Polymerase Mix | 2 μl | |
Total volume | 20 μl |
Please note: Reagents for the following steps are not provided within this kit.
DNA template removal (optional)
Depending on the down-stream application, removal of template DNA might be required. We recommend a salt-resistant, high efficiency DNAase such as Turbo™DNAse (ThermoFisher). Follow the manufacturer instructions.
RNA purification
Purification of RNA is required for certain applications such as measurement of Digoxigenin-labelled RNA probe concentration. Spin column purification will remove proteins, salts and unincorporated nucleotides. Please follow the manufacturer instructions and ensure that the columns match with product size and possess a sufficient binding capacity (e.g. RNA Clean & Concentrator™ columns (Zymo Research) or Monarch® RNA Cleanup kit (NEB)). Other RNA purification methods such as LiCl precipitation may work but have not been tested.
RNA quantitation
RNA concentration can be determined by absorbance measurement at 260 nm (A260) according to the Law-of-Lambert-Beer (A260 = 1 correspond to 40 μg/ml ssRNA).
Related products: Digoxigenin-11-UTP, #NU-821-DIGX
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