Day 1
[Prepare reagents]
- cold ethanol – on ice
- 5 M NaOH in DEPC water
- 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 1.5 mL volume microfuge tube, add 75 µL Nuclease-free water, 15 µL 3M sodium acetate, and the 60 µL of the in vitro transcription reaction mixture. Total volume of the reaction mixture will be µL.
- 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.)
- Transfer the aqueous layer to a new 500 µL 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.
- Precipitate the RNA by adding 0.1 volume of 3 M sodium acetate and 2.5 volume of cold ethanol, and mixing well. Let the precipitation to go overnight at -20˚C.
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% 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.