The typical recovery rate of the RNA standard transcription system is lugRNA/ug plasmid DNA, which can be increased to 5~l0ug RNA/ug plasmid DNA by using the following method. large quantities of prepared RNA can be used for in vitro translation.

Operation method

RNA bulk transcription synthesis

Materials and Instruments

Template DNA or plasmid
TE TE Saturated Phenol Chloroform Isonitol 3mol LNaAc Anhydrous Ethanol 5X Transcription Buffer lOOmmoL LDTT RNasin rATP rGTP rUTP rCTP SF6 T3 or T7 RNA Polymerase Isoamyl Alcohol DNase 7.5mol L Ammonium Acetate Anhydrous Ethanol Ethanol

Move

I Materials and equipment

1) Template DNA or plasmid

2) TE

3)TE Saturated Phenol: Chloroform

4) Chloroform: Isolactone (24:1)

5) 3mol/LNaAc (pH 5.2)

6) Anhydrous ethanol

7) 5X transcription buffer: 200 mmol/L Tris-HCl (pH 7.9), 30 mmol/L MgCl2, 10 mmol/L spermidine, 50 mmol/L NaCl.

8)lOOmmoL/LDTT

9)RNasin

10)rATP,rGTP,rUTP,rCTP, each 2.5 mmoI/L

11)SF6,T3 or T7 RNA polymerase

12)TE-saturated phenol:chloroform:isoamyl alcohol (25:24:1, PH4.5)

13)DNase

14)7.5mol/L ammonium acetate

15) anhydrous ethanol

16)70% ethanol

Methods


1) Add the following components in sequence at room temperature:

5X Transcription Buffer 2ul

DTT, 100 mmol/L 10ul

RNasin 100U

rATP,rGTPT,rCTP,rUTP (2.5 mmol/L each) 20ul

Linear template DNA(1~2.5 mg/ml) 2ul

SP6, T3 or T7 RNA polymerase 40U

Add water without RNAase to a final volume of l00ul.

2) React at 37~40℃ for 1 h.

3) Remove the DNA template as described in RNA Standard Transcription Reaction, and purify the RNA transcript.

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