Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit Troubleshooting

Multiple reaction

products,

transcripts of the

wrong size1.Reaction products produce a smear when run on a denaturing gelIf the RNA appears degraded (e.g. smeared), remove residual RNase from the DNA template preparation before in vitro transcription. Do this by digesting the DNA prep with proteinaseK(100–200µg/mL) in the presence of 0.5% SDS for 30 18min at 50°C, follow this with phenol/chloroform extraction. The RNase Inhibitor that is present in the transcription reaction, can only inactivate trace RNase contamination. Large amounts of RNase contamination will compromise the size and amount of transcription products. 2.Reaction products run as more than one band, or as a single band smaller than expecteda.Sample is not adequately denatured in the gelIf the amount of RNA produced is acceptable, but the size of the product is unexpected, consider that the RNA may be running aberrantly due to secondary structure. Sometimes the RNA will run as two distinct bands on a native agarose gel, but when the same RNA is run on a denaturing gel, it will migrate as a single band of the expected size.b.Premature termination of transcriptionIf denaturing gel analysis shows the presence of multiple bands or of a single band smaller than the expected size, there may be problems with premature termination by the polymerase. Possible causes of this are sequences which resemble the phage polymerase termination signals, stretches of a single nucleotides, and GC-rich templates. Different phage polymerases recognize different termination signals, so using a different polymerase promoter may help. Termination at single polynucleotide stretches can sometimes be alleviated by decreasing the reaction temperature (Krieg, P.A. 1990). We suggest testing 30°C, 20°C and 10°C. However, decreasing the reaction temperature will also significantly decrease the yield of the reaction. There is a report that single-stranded binding (SSB) protein increased the transcription efficiency of a GC rich template (Aziz and Soreq, 1990). 3.Reaction products are larger than expecteda.Persistent secondary structuremMESSAGE mMACHINE® products occasionally run as 2bands; 1larger than the expected size, and 1 at the expected size. This may occur with transcripts from the pTRI-Xef control template, even when the RNA is denatured during the electrophoresis. This phenomenon occurs because of persistent secondary structure. To verify this, the band that migrates at the expected size can be excised from the gel and run in a second denaturing gel. If the RNA runs as a doublet in the second gel also, it is a good indication that the larger band is simply an artifact of electrophoresis.b.Circular templateLonger-than-expected transcription products will be seen if any of the template molecules are circular. This is typically caused by incomplete digestion of a plasmid template. Since the RNA polymerases are extremely processive, even a small amount of circular template can produce a large amount of RNA.mMESSAGE mMACHINE® Kit User Guide