Preparation of randomly AF488-modified RNA probes by in vitro transcription with UTP-PEG5-AF488
Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
RNT-101-AF488 | 20 reactions x 20 μl | 421,60 | 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
Spectroscopic Properties: λexc 494 nm, λem 515 nm, ε 73.0 L mmol-1 cm-1 (Tris-HCl pH 7.5)
Description:
HighYield T7 AF488 RNA Labeling Kit (UTP-based) is designed to produce randomly AF488-modified RNA probes via in vitro transcription. Such probes are ideally suited for in situ hybridization and Northern Blot experiments. AF488 (also known as Alexa Fluor® 488) is a hydrophilic dye with increased photostability compared to Fluorescein.
UTP-PEG5-AF488 is efficiently incorporated into RNA as substitute for its natural counterpart UTP using an optimized reaction buffer and T7 RNA Labeling Polymerase Mix. 35 % UTP-PEG5-AF488 substitution typically results in an optimal balance between reaction and labeling efficiency. Individual optimization of UTP-PEG5-AF488/UTP ratio however, can easily be achieved with the single nucleotide format. The resulting AF488-modified RNA probe can subsequently be detected by fluorescence spectroscopy.
The kit contains sufficient reagents for 20 labeling reactions of 20 μl each (35 % UTP-PEG5-AF488 substitution, 2.5 mM ATP, GTP, CTP, 0.2 mM UTP, 0.1 mM UTP-PEG5-AF488).
A 20 μl reaction yields about 5-7 μg RNA after 30 min incubation (1 μg T7 control template, 1.4 kb RNA transcript). Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation).
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)
UTP-PEG5-AF488
1x 10 μl (5 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% UTP-PEG5-AF488 substitution following the standard protocol below however, individual optimization might improve results for individual applications (e.g. variation of UTP-PEG5-AF488/UTP ratio between 30 - 50 %).
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) | 5 μl | 2.5 mM |
10 mM UTP working solution (s. 2.2) | 0.4 μl | 0.2 mM |
5 mM UTP-PEG5-AF488 | 0.4 μl | 0.1 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 AF488-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.
Total 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).
Incorporation rate of fluorophore
The efficiency of RNA labeling can be estimated by calculating the ratio of incorporated fluorophores to the number of bases (dye / base).
[Please note: Blanc correction with probe buffer solution is required.]
1. Measurement of the nucleic acid-dye conjugate absorbance:
Measure the absorbance of the labeled RNA fragment at 260 nm (A260) and at the excitation maximum (λexc)of dye (Adye).
2. Correction of A260 reading:
To obtain an accurate nucleic acid absorbance measurement, the contribution of the dye at 260 nm needs to be corrected. Use the following equation:
Abase = A260 - (Adye x CF260)
Correction Factor for AF488: CF260 = 0.3
3. Calculation of dye to base ratio by the law of Lambert-Beer (A = c x ε x d):
dye/base ratio = (Adye x εbase) / (Abase x εdye)
Extinction coefficients:
AF488: εdye = 73,000 cm-1 M-1
ssRNA: εbase = 12,030 cm-1 M-1 (average, 50% GC)
3. Calculation of the degree of labeling (DOL)
The degree of labeling (DOL) indicates the number of dyes per 100 bases.
DOL = 100 x dye/base ratio
Example: A dye/base ratio of 0.02 corresponds to a DOL of 2 that corresponds to 2 dyes per 100 bases.
Related products: Aminoallyl-UTP-PEG5-AF488, #NU-821-PEG5-AF488
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