Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure

If the reaction is trace-labeled:

After the incubation (before or after TURBO DNase treatment), remove an aliquot of trace-radiolabeled reactions to assess yield by TCA precipitation (see step5., “Quantitation by trace radiolabeling” on page 14).

5.(optional) Add 1µL TURBO DNase, mix well and incubate 15min at 37°CThis DNase treatment removes the template DNA. For many applications it may not be necessary because the template DNA will be present at a very low concentration relative to the RNA.

a.Add 1µL TURBO DNase, and mix well.b.Incubate at 37°C for 15min.

The degree of purification required after the transcription reaction depends on what will be done with the RNA. Four different methods follow, choose one or more according to your application and resources.

1.MEGAclear Kit

The MEGAclear Kit was developed specifically for purifying RNA from high

yield in vitro transcription reactions. The quick and simple procedure removes nucleotides, short oligonucleotides, proteins, and salts from RNA. The RNA

recovered can be used for any application that requires high purity RNA.

2.Lithium chloride precipitation

Lithium Chloride (LiCl) precipitation is a convenient and effective way to remove unincorporated nucleotides and most proteins. Lithium chloride precipitation, however, does not precipitate transfer RNA and may not efficiently precipitate RNAs smaller than 300nucleotides. Also, the concentration of RNA should be at least 0.1 µg/µL to assure efficient precipitation. To precipitate from mMESSAGE mMACHINE® reactions that are thought to have relatively low yields of RNA, do not dilute the transcription reaction with water prior to adding the LiCl

Precipitation Solution the first substep below.

a.Stop the reaction and precipitate the RNA by adding 30µL Nuclease-free

Water and 30µL LiCl Precipitation Solution.

b.Mix thoroughly. Chill for ≥30min at –20°C.

c.Centrifuge at 4°C for 15min at maximum speed to pellet the RNA.

d.Carefully remove the supernatant. Wash the pellet once with ~1mL 70%

ethanol, and re-centrifuge to maximize removal of unincorporated

nucleotides.

e.Carefully remove the 70% ethanol, and resuspend the RNA in a solution or

buffer appropriate for your application. Determine the RNA concentration

and store frozen at –20°C or –70°C.

3.Spin column chromatography

Spin columns will remove unincorporated nucleotides, including unincorporated cap analog that may inhibit in vitro translation.

Life Technologies offers several products for RNA storage, these include: Nuclease-free Water (not DEPC-treated): Cat. nos.THE RNA Storage Solution: Cat. nos.AM9930–AM9939

TE Buffer: Cat. nos.0.1mM EDTA: Cat. no.AM9860, AM9861AM7000, AM7001

AM9912

mMESSAGE mMACHINE® Kit User GuideRecovery of the RNA12