Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure

Prepared spin columns such as NucAway Spin Columns can be used by

following the manufacturer’s instructions. Alternatively, instructions for

preparing spin columns are given in section“Spin column preparation and use” on page 22.

4.Phenol:chloroform extraction and isopropanol precipitation

This is the most rigorous method for purifying transcripts. It will remove all enzyme and most of the free nucleotides from mMESSAGE mMACHINE® Kit

reactions. Since the RNA is precipitated, this method can also be used for buffer exchange.a.Add 115µL Nuclease-free Water and 15µL Ammonium Acetate Stop Solution, and mix thoroughly.

b.Extract with an equal volume of phenol/chloroform (it can be water-

saturated, buffer-saturated, or acidic), and then with an equal volume of

chloroform. Recover aqueous phase and transfer to new tube.

c.Precipitate the RNA by adding 1volume of isopropanol and mixing well.

d.Chill the mixture for at least 15min at –20°C. Centrifuge at 4°C for 15min at

maximum speed to pellet the RNA. Carefully remove the supernatant

solution and resuspend the RNA in a solution or buffer appropriate for

your application.

e.Store frozen at –20°C or –70°C.

Quantitation of

reaction products1.Quantitation by UV light absorbanceReading the A260 of a diluted aliquot of the reaction is clearly the simplest way to

determine yield, but any unincorporated nucleotides and/or template DNA in the mixture will contribute to the reading. Typically, a 1:100 dilution of an aliquot of a mMESSAGE mMACHINE® reaction will give an absorbance reading in the linear range of a spectrophotometer.

For single-stranded RNA, 1 A260 unit corresponds to 40µg/mL, so the RNA yield can be calculated as follows:

A260 x dilution factor x 40 = µg/mL RNA

2.Assessing RNA yield with RiboGreen® assay

If you have a fluorometer, or a fluorescence microplate reader, Molecular Probes’ RiboGreen® fluorescence-based assay for RNA quantitation is a convenient and sensitive way to measure RNA concentration. Follow the manufacturer’s

instructions for using RiboGreen.

3.Quantitation by ethidium bromide fluorescence

The intensity of ethidium bromide staining can be used to get a rough estimation of the RNA yield.

Ethidium bromide spot assay

If unincorporated nucleotides have been removed, an ethidium bromide spot

assay can be used to quantitate RNA concentration. Make a standard curve with several 2-fold dilutions of an RNA solution of known concentration. Start at about 80ng/µL, and go down to about 1.25ng/µL. Make a few dilutions of the unknown RNA, and add ethidium bromide to 1ng/µL to each dilution of both RNAs. Spot 2µL of the standard curve RNA samples and the unknown RNA dilutions onto

mMESSAGE mMACHINE® Kit User Guide13