1.5 Troubleshooting
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1. overnight by autoradiography The SP6 polymerase 10 u uL stock will sometimes freeze if the freezer is much below 20 C Adjust freezer temperature to 206C or keep a more concentrated stock2. 1 5 Troubleshooting This is a highly reproducible and reliable approach to making large quantities of RNA As long as your DNA is clean phenol extracted and ethanol precipitated miniprep DNA and the enzymes haven t been abused e g left out on the bench etc all should go well When using miniprep DNA remember you must omit the RNAse step or any RNA you synthesize will get chewed up Keep all of your key reagents e g 4NTP cap cap 0 1 M DTT etc in small aliquots frozen at 80 6C and work off of the same aliquot each time putting a dot with magic marker on the side of the tube each time it s thawed and refrozen That way you have some idea how many times its been used When a problem develops you can pull out a fresh unused aliquot of everything and be confident that they should work If not suspect any newly prepared reagent or get give transcript from to someone else If the problem is corrected it suggests a problem with either your reagents or your assays for expression Transcription reaction mRNA products may be quantitated by fluorimetry see appropriate section of lab manual Radiolabelled RNA can also be produced by incorporating a nucleotide labelled with 32P or 35S at the position For a reasonable signal 0 5 uL of a 35S UTP 10 mCi mL in a 10 uL reaction is sufficient The 5X NTP mix will not have to be adjusted 1 uL of this plus 9 uL of deionized formamide can be run on an 10 acrylamide 8 M urea TBE gel and visualized
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