Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure

plastic wrap placed on a UV transilluminator. Compare the fluorescence of the RNAs to estimate the concentration of the unknown RNA sample. Make sure that the sample dilutions are in the linear range of ethidium bromide fluorescence. This assay will detect as little as 5ng of RNA with about a 2-fold error.

Denaturing gel electrophoresis

If unincorporated nucleotides have not been removed from the reaction, an

aliquot of the mMESSAGE mMACHINE® reaction should be run on a denaturing agarose or acrylamide gel alongside an aliquot of an RNA of known

concentration. See section“Additional procedures” on page 19 for instructions on running gels. Stain the samples with ethidium bromide, and simply compare the intensity of the unknown sample to the known RNA to estimate its concentration.4.Agilent® Bioanalyzer® system and RNA LabChip® Kits

RNA can be evaluated on an Agilent® 2100 Bioanalyzer® using one of their RNA LabChip® Kits to get an idea of what percentage of the transcription products are full-length. Follow the manufacturer’s instructions for using the bioanalyzer and the RNA LabChip® Kit.

5.Quantitation by trace radiolabeling

TCA precipitation

If a trace amount of radiolabel was included in the mMESSAGE mMACHINE®

reaction, it can be used to determine yield. First precipitate with TCA to determine the proportion of radiolabel that was incorporated into RNA. (TCA will precipitate nucleic acids as small as 18nt.)

a.Dilute 1µL of the completed mMESSAGE mMACHINE® reaction with 1µL TE Buffer in a nuclease-free 1.5mL microfuge tube, and vortex thoroughly to

ensure that the newly synthesized RNA is completely solubilized.

b.Add 150µL of carrier DNA or RNA (1mg/mL) (Sheared Salmon Sperm

DNA Cat. no.AM9680 can be used for this) to the diluted reaction products,

and mix thoroughly.

c.Transfer 50µL of the RNA + carrier nucleic acid mixture to aqueous

scintillation cocktail and count in a scintillation counter. This will measure

the total amount of radiolabel present in the reaction mixture

(unincorporated and incorporated counts).

d.Transfer another 50µL of the RNA + carrier nucleic acid mixture to a

12x75mm glass tube, and add 2mL of cold 10%TCA (trichloroacetic acid).

Mix thoroughly and place on ice for 10min. This will precipitate nucleic

acids, but not free nucleotides.

e.Collect the precipitate via vacuum filtration through a Whatman® GF/C

glass fiber filter (or its equivalent).

f.Rinse the tube twice with 1mL of 10% TCA and then rinse once with 3–5mL of 95% ethanol. Pass each of the rinses through the GF/C filter.

g.Place the filter in a scintillation vial, add aqueous scintillation cocktail, and count in a scintillation counter. This will give the TCA precipitated counts

(radiolabel that was incorporated into RNA).

h.Divide the cpm in Step g. by the cpm in Step c. to determine the fraction of label incorporated into RNA (multiply by 100 for percent incorporation).

mMESSAGE mMACHINE® Kit User Guide14