protocol:rna_invitro

Table of Contents

Order template
in vitro transcription
- 1. Annealing Reaction
- 2. in vitro Transcription
Purify RNA
- Option 1: PAGE

Order template

in vitro transcription

1. Annealing Reaction

Dilution: dilute each DNA strand to a final concentration of 100 µM with water. (# nmol X 10 = µL of water to add)
Make the following cocktails in an Eppendorf tube (scale it up according to your final reaction volume)

Volume (30 uL in total)	Reagent
7.5 uL	DNA strand 1
7.5 uL	DNA strand 2
9 uL	10 mM MgCl2
6 uL	H2O

Incubate the annealing reactions at 95.0 ºC for 5 min, and then immediately put it on ice.

2. in vitro Transcription

Mix the following reagents in a 50 mL falcon tube
Note
- SP: Spermidine; PEG: Polyethylene glycol
- RNAP: T7 RNA polymerase (5000 unit)

TOTAL	H2O	0.5M Tris	0.25M MgCl2	0.25M DTT	0.25M SP	PEG	ATP	GTP	CTP	UTP	DNA	RNAP
50 uL w/ PEG	13.8 uL	10 uL	6 uL	6 uL	0.4 uL	4 uL	1.5 uL	1.5 uL	1.5 uL	1.5 uL	0.8 uL	3 uL
50 uL w/o PEG	17.8 uL	10 uL	6 uL	6 uL	0.4 uL	0 uL	1.5 uL	1.5 uL	1.5 uL	1.5 uL	0.8 uL	3 uL
9 mL	3204 uL	1800 uL	1080 uL	1080 uL	72 uL	0 uL	270 uL	270 uL	270 uL	270 uL	144 uL	540 uL
12 mL	4272 uL	2400 uL	1440 uL	1440 uL	96 uL	0 uL	360 uL	360 uL	360 uL	360 uL	192 uL	720 uL
15 mL	5340 uL	3000 uL	1800 uL	1800 uL	120 uL	0 uL	450 uL	450 uL	450 uL	450 uL	240 uL	900 uL

Another：

TOTAL	5XTRB	T7 RNAP	50μM DNA	rNTPs	DEPC water
50μL	10μL	1.5μL	1.5μL	1.5μL	35.5μL

5XTRB(Transcription Buffer)：

5x 浓度	母液体积	母液浓度	DEPC water
200mMTris-HCl pH8.0	8 mL	1 M	—
10mM SP	1.6 mL	0.25 M	—
5mM DTT	0.2 mL	1 M	—
MgCl2梯度:	—	—	—
50mM	2 mL	1 M	28.2 mL
75 mM	3 mL	1 M	27.2 mL
100 mM	4 mL	1 M	26.2 mL
150 mM	6 mL	1 M	24.2 mL
200 mM	8 mL	1 M	22.2 mL

Purify RNA

Option 1: PAGE

Choose proper concentration of PAGE

8% Acrylamide: 60~400bp
12% Acrylamide: 40~200bp
15% Acrylamide: 25~150bp
20% Acrylamide: 6~100bp

Denatured PAGE Gel

500V,130mA at 4C for 6~7 hours

Crush & Soak

Cut RNA band from the gel under 260nm wavelength fluorescence light
Crush the gel band into fine grains by using 50mL Falcon tube and 50mL syringe
Soak in 2 volumes of CSB (Crush&Soak buffer) for 6 hours at room temperature, and collect solution by filtering it
Repeat the step above
Ethanol Precipitate the RNA from the collected supernatant (or run the supernatant through desalting column)