Life Ambion T7启动子转录试剂盒说明书

A

Additional procedures

Analysis of

transcription

products by gel

electrophoresisSupplemental Information1.Agarose or Acrylamide?The size of mMESSAGE mMACHINE reaction products can be assessed by running an aliquot of the reaction on an agarose or polyacrylamide gel. Transcripts larger than about 1.5kb should be run on agarose gels, whereas

polyacrylamide gels (4–5%) are better for sizing smaller transcripts. Since

secondary structure in the transcript may cause aberrant migration and/or

multiple bands, the gel should be run under denaturing conditions. For agarose gels, this means glyoxal or formaldehyde gels, prepared and run according to standard procedures (Molecular Cloning, A Laboratory Manual, 1989).

Instructions for preparing and running denaturing acrylamide gels are supplied in section“Denaturing acrylamide gel mix” on page 25.

2.Sample preparation

To get good resolution of the RNA, load ~1µg per gel lane. For denaturing

polyacrylamide gels add an equal volume of Gel Loading BufferII to each

sample, and heat for 3–5min at 80–90°C. (Gel Loading Buffer II cannot be used with glyoxal agarose gels and it will not completely denature samples run on formaldehyde agarose gels. Use a loading buffer specifically formulated for the type of agarose gel you plan to run.)

To stain the RNA with ethidium bromide during electrophoresis do one of the following:

a.Add 0.5µg/mL ethidium bromide to the gel mix

b.Add 0.5µg/mL ethidium bromide to the running buffer

c.Add 10µg/mL ethidium bromide to the RNA samples (and gel loading

buffer) before loading the gel.

(Because single-stranded nucleic acids bind ethidium less efficiently than double-stranded nucleic acids, the fluorescence of RNA samples on a denaturing agarose gel will be less intense than the same amount of DNA.)

3.Visualizing reaction products

a.Ethidium bromide stained samples

View ethidium bromide stained gels on a UV transilluminator. Ideally there

will be a single, tight band at the expected molecular weight. See

section“Multiple reaction products, transcripts of the wrong size” on page

18 for troubleshooting suggestions if this is not what appears on your gel.

b.Radioactively-labeled transcripts

mMESSAGE mMACHINE® Kit User Guide19