dsRNA synthesis for RNAi applications via T7 RNA Polymerase-mediated in vitro transcription
Cat. No. | Amount | Price (EUR) | Buy / Note |
---|---|---|---|
RNT-134 | 50 reactions x 20 μl | 265,00 | 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 after date of delivery
Description:
HighYield T7 RNAi Kit is designed to produce large amounts of double-stranded RNA (dsRNA) > 200 bp via T7 RNA Polymerase-mediated in vitro transcription. The resulting dsRNA can subsequently be used for RNA interference (RNAi) applications. Preparation of small interfering dsRNA, known as siRNA (approximately 20 bp long) is principally feasible as well however, we recommend a chemical siRNA synthesis approach for short dsRNAs.
The kit contains sufficient reagents for 50 reactions of 20 μl each (7.5 mM each NTP). A 20 μl reaction yields > 40 μg dsRNA (> 2 mg/ml) after 30 min incubation (1 μg each T7 control template I & II, 0.5 kbp RNA transcripts). Yields may however vary depending on the template (promotor design, sequence length, secondary structure formation).
Content:
HighYield T7 RNA Polymerase Mix
3x 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)
PCR-grade water
1x 1.2 ml
DTT
1x 150 μl (100 mM)
DNase I Solution
1x 60 μl (1 u/μl)
RNase A Solution
1x 200 μl (4 mg/ml)
Sodium Acetate Solution
1x 1 ml (3 M)
T7 G-initiating control template I (0.5 kbp)
10 μl (0.5 μg/μl)
T7 G-initiating control template II (0.5 kbp)
10 μl (0.5 μg/μl)
To be provided by user
T7 Promotor-containing DNA template
Isopropanol
70 % Ethanol
Important Notes (Read before starting)
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
Template requirements
Minimum T7 promotor sequences:
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'-TAATACGACTCACTATTANN…-3'
Bold: First base incorporated into RNA, NN: ideally GG
Option 1:
Prepare two identical DNA templates with a single T7 promoter at opposite ends of the region to be transcribed. In vitro transcription reactions of each template can be performed to obtain complementary ssRNA molecules. An additional annealing step is required. Alternatively, transcribe both templates simultaneously in one reaction. dsRNA and unspecific aggregates are most often directly generated in this case. Aggregate formation can be solved by performing an additional denaturation and annealing step.
Option 2:
Prepare one DNA template with opposite T7 promoter at the 5' ends of each strand. dsRNA and unspecific aggregates are most often directly generated during in vitro transcription. Aggregate formation can be solved by performing an additional denaturation and annealing step.
In vitro Transcription protocol
The protocol is optimized for 1 μg DNA template (refer to "Important Notes" regarding template requirements).
Component | Volume | Final conc. |
PCR-grade water | X μl | |
HighYield T7 Reaction Buffer (10x) | 2 μl | 1x |
DTT (100 mM) | 2 μl | 10 mM |
ATP (100 mM) | 1.5 μl | 7.5 mM |
UTP (100 mM) | 1.5 μl | 7.5 mM |
CTP (100 mM) | 1.5 μl | 7.5 mM |
GTP (100 mM) | 1.5 μl | 7.5 mM |
Template DNA | X μl | 1 μg |
HighYield T7 RNA Polymerase Mix | 2 μl | |
Total volume | 20 μl |
Annealing/dsRNA preparation
An annealing step is recommended independent of the template strategy. Successfull annealing depends on nearly equimolar amounts of ssRNA. If ssRNA from separate in vitro transcription reactions are annealed (see „Template strategies, Option 1“), first check on transcription efficiency of each ssRNA by agarose gelelectrophoresis.
DNA template and ssRNA removal
dsRNA Purification
RNA quantitation
RNA concentration can be determined by absorbance measurement at 260 nm (A260) according to the Law-of-Lambert-Beer (A260 = 1 corresponds to 40 μg/ml dsRNA).
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