Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit

mMESSAGE mMACHINE® Kit Procedure

Plasmid templatesDNA should be relatively free of contaminating proteins and RNA. We observe the greatest yields with very clean template preparations. Most commercially available plasmid preparation systems yield DNA that works well in the mMESSAGE

mMACHINE® Kit. Otherwise, a DNA miniprep procedure that generally yields high quality template is presented in section“Miniprep for isolating transcription-quality plasmid DNA” on page 23.LinearizationPlasmid DNA must be linearized with a restriction enzyme downstream of the insert to be transcribed. Circular plasmid templates will generate extremely long, heterogeneous RNA transcripts because RNA polymerases are very processive. It is generally worthwhile to examine the linearized template DNA on a gel to confirm that cleavage is complete. Since initiation of transcription is one of the limiting steps of in vitro transcription reactions, even a small amount of circular plasmid in a template prep will generate a large proportion of transcript.

Although we routinely use all types of restriction enzymes, there has been one report of low level transcription from the inappropriate template strand in plasmids cut with restriction enzymes leaving 3' overhanging ends (produced by KpnI, PstI, etc.; Schendorn and Mierindorf, 1985).

After linearization

Terminate the restriction digest by adding the following:

1/20thvolume 0.5M EDTA

1/10thvolume of 3M Naacetate or5MNH4acetate

2volumes of ethanol

Mix well and chill at –20°C for at least 15min. Then pellet the DNA for 15min in a microcentrifuge at top speed. Remove the supernatant, re-spin the tube for a few seconds, and remove the residual fluid with a very fine-tipped pipet. Resuspend in dH2O or TE buffer at a concentration of 0.5–1µg/µL.

ProteinaseK treatment

Note that DNA from some miniprep procedures may be contaminated with residual RNaseA. Also, restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When transcription from a template is suboptimal, it is often helpful to treat the template DNA with proteinaseK (100–200µg/mL) and 0.5%SDS for 30min at 50°C, follow this with phenol/chloroform extraction (using an equal volume) and ethanol precipitation.

PCR templates

DNA generated by PCR can be transcribed directly from the PCR provided it contains an RNA Polymerase promoter upstream of the sequence to be transcribed. PCR products should be examined on an agarose gel before use as a template in

mMESSAGE mMACHINE® to estimate concentration, and to verify that the products are unique and the expected size.

mMESSAGE mMACHINE® Kit User Guide9