Life Ambion T7启动子转录试剂盒说明书

mMESSAGE mMACHINE® Kit Troubleshooting

Troubleshooting

low yield

162.Expected yield from the control reactionThe yield from the control reaction for T7 and T3 Kits should be 20–30µg of RNA, and 15–25µg with the SP6 Kits. If a [32P]NTP was added to the transcription reaction as a tracer, approximately 15% of the radiolabel should be incorporated.3.What to do if the control reaction doesn’t work as expectedIf the yield of RNA from the control reaction is low, something may be wrong either with the procedure or the kit, or the quantitation is in error. a.Double check the RNA quantitationTo confirm that the quantitation is correct, verify the yield by an independent method. For example if TCA precipitation was used to assess yield, try also running an aliquot of the reaction on an agarose gel. b.Try the positive control reaction againIf the yield is indeed low by two different measurements, there may be a technical problem with the way the kit is being used. For example, the spermidine in the 10X Reaction Buffer may cause precipitation of the template DNA if it is not diluted by the other ingredients prior to adding the DNA. (This is the reason that the water is added first.) Repeat the reaction, following the procedure carefully. If things still don’t go well, contact Technical Services for more ideas.The amount of RNA synthesized in a standard 20µL mMESSAGE mMACHINE® reaction should be 15–20µg and may exceed 30µg; however, there is a great deal of variation in yield from different templates. If the yield is low, the first step in troubleshooting the reaction is to use the pTRI-Xef control template in a standard mMESSAGE mMACHINE® reaction. 1.Neither my template nor the control reaction worksDouble check that you have followed the procedure accurately, and consider trying the control reaction a second time. If the kit control still doesn’t work, it is an indication that something may be wrong with the kit, call our Technical Support group for more ideas.2.The control reaction works, but my template gives low yieldIf the transcription reaction with your template generates full-length, intact RNA, but the reaction yield is significantly lower than the amount of RNA obtained with the pTRI-Xef control template, it is possible that contaminants in the DNA are inhibiting the RNA polymerase. A mixing experiment can help to differentiate between problems caused by inhibitors of transcription and problems caused by the sequence of a template. Include three reactions in the mixing experiment, using the following DNA templates:1. 1µL pTRI-Xef control template2.experimental DNA template (0.5µg plasmid or 2–6µL PCR product)3.a mixture of 1 and 2Assess the results of the mixing experiment by running 0.5–1µL of each transcription reaction on a denaturing gel as described in section , “Additional procedures” on page 19.mMESSAGE mMACHINE® Kit User Guide