Standard RNA Synthesis (E2050)
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**** If you need to do in vitro transcription, elute your DNA in DEPC water!!!! The Template DNA needs to be in DEPC water.
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Reactions are typically 20 μl but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.
- Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Keep on ice.
- Assemble the reaction at room temperature in the following order:
Reaction set up for short transcripts (< 0.3 kb):
| Nuclease-free water | X µl |
| NTP Buffer Mix | 10 µl (6.7 mM each NTP final) |
| Template DNA | X µl (1 µg) |
| T7 RNA Polymerase Mix | 2 µl |
| Total reaction volume | 30 µl |
3. Mix thoroughly and pulse-spin in a microfuge. For reactions with short RNA transcripts (< 0.3 kb), we recommend an incubation time of 4 hours or longer. It is safe to incubate the reaction for 16 hours (overnight). For example, we have achieved good yield with only 0.2 μg plasmid template encoding a 50-mer RNA by incubating the reaction overnight at 37°C.
***Reaction time depends on template amount, quality and RNA transcript length. For reactions with transcripts longer than 0.3 kb, 2 hour incubation should give you the maximum yield. For reaction times of 60 minutes or less, a water bath or heating block may be used; for reaction times longer than 60 minutes, we recommend using a dry air incubator or a thermocycler to prevent evaporation of the sample.
***The kit contains sufficient materials for 50 reactions.
***Note that the amount of NTP Buffer Mix in a standard 20 μl reaction can vary from 2 to 10 μl. The final yield is proportional to the amount of input nucleotides, meaning that the nucleotide incorporation efficiency remains the same when different amounts of NTP are used. Figure 1 shows the time course of standard RNA synthesis from 1 μg control DNA template coding for a 1.8 kb RNA transcript with the HiScribe T7 Quick Kit using 10 μl and 5 μl NTP Buffer Mix in a 20 μl reaction.
Figure 1. RNA synthesis with different amounts of NTP.
Reactions were incubated at 37°C in a thermocycler. Transcripts were purified by spin columns and quantified on a NanoDrop™ Spectrophotometer.
4. Optional: DNase treatment to remove DNA template. Standard reactions are capable of generating large amounts of RNA, at concentrations up to 10 mg/ml. As a result, the reaction mixture is quite viscous. It is easier to perform DNase treatment after the reaction mixture is diluted. To remove template DNA, add 30 μl nuclease-free water to each 20 μl reaction, followed by 2 μl of DNase I (RNase-free), mix and incubate for 15 minutes at 37°C.
| DEPC water | 22 μl |
| Reaction Mixture | 30 μl |
| DNase I | 6 μl |
| 10X DNase I buffer | 2 μl |
| Total Volume | 60 μl |
5. Quantification
Day 1
[Prepare reagents]
- cold ethanol – on ice
- 5 M NaOH in DEPC water (needed when making 3M sodium acetate)
- 3 M sodium acetate – prepare using glacial acetic acid (17.4 M) and DEPC water-based 5M NaOH
(Adding water to NaOH is an exothermic process. So, add NaOH pallets to the water in small amounts. Add more after you confirmed that the added pallets are completely dissolved.)
[Phenol:Chloroform Extraction]
- In 2.0 mL volume microfuge tube, add:
| DEPC water | 75 µL |
| 3M sodium acetate | 15 µL |
| in vitro transcription reaction mixture | 60 µL |
| Total volume | 150 µL |
2. Extract with an equal volume of phenol : chloroform : isoamyl alcohol (25 : 24 : 1). Recover the aqueous layer on top. (*For extractions, do not retrieve all of the aqueous layer. It’s better to lose some material then having the risk of contamination.
3. Transfer the aqueous layer to a new 1.5 mL volume microfuge tube. Then extract with an equal volume of chloroform. Recover the aqueous layer on top, and transfer to a new 1.5 mL volume microfuge tube.
4. Precipitate the RNA by adding 2.5 volume of cold ethanol, and mixing well. Let the precipitation to go overnight at -20˚C.
**For later precipitation for RNA storage also add 0.1 volume of 3M sodium acetate.
Day 2
[Prepare reagents]
- 3 L of DEPC water
- 1M Tris-HCl (pH 7.5) in DEPC water
- 0.5 M EDTA in DEPC water
- 10X TBE in DEPC water
- 5X DNA loading Dye in DEPC water
[Handling of the Precipitated RNA Pellet]
- Retrieve the tube from -20˚C.
- Carefully remove the supernatant solution and dry the RNA pellet on thermomixer: 37˚C, at 700 rpm for 20 min.
- Do not resuspend the RNA in DEPC-water, until you are ready to check the RNA products on the 0.8% DEPC-water based agarose gel.
[Quantification: NanoDrop / Agarose Gel Electrophoresis]
- Measure the RNA concentration on the NanoDrop – try to aim for A260/A280 at around 1.8.
- Run the denaturing Agarose Gel Electrophoresis.
- Prepare 1.0% or 1.5% agarose gel in DEPC water, and pre-stain the gel with SyberGold dye (1:20,000).
| Ladder | |
| Ladder | 20 µL |
| 10X TBE in DEPC water | 5 µL |
| Formamide | 37.5 µL |
| RNA Sample | |
| RNA product | 8 µL |
| 10X TBE in DEPC water | 2 µL |
| Formamide | 15 µL |
- Heat at 70 ˚C for 10 min; Chill on ice for 5 min.
- Run the gel in 0.5X TBE in DEPC water.