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Extra resources for A Laboratory Guide to In Vitro Transcription
Nevertheless, for preparative purposes, spinning just once, and for only 40 minutes seems to be adequate, since further chromatographic purifications will be introduced later. 5-ml cushions can be used, so that a little more sample can be loaded per SW27 tube. 5 mM PMSF and 1 % Trasylol. 1. 5 ml per pad. The pads should be done as before, that is, with regular homogenization buffer containing only 2 M sucrose, 10% glycerol and no low fat milk. 4 M sucrose pad. > Proceed with the dissection as before, but do not mince the tissue.
With a hand-held Geiger counter, localize the position of the unincorporated UTP (strong band just above the BPB). Using the edge of the glass plate not adhered to the gel, cut this portion out. This step mainly serves to reduce contamination of the fixing solution and gel drier. > Put the gel and the glass plate into a tray containing 20% ethanoV10% acetic acid. Fix for 15-20 minutes. > Remove the glass plate and gel from the fixer, carefully drain by letting it stand almost vertically for a few seconds, and then transfer the gel to a dry piece ofWhatmann 3 MM paper.
Collect approximately 4 ml and discard. 7% agarose). 5 M. > Extract 3 times with phenol/chloroform. 5 volumes of ethanol. Leave in dry ice for 20 minutes. 1. Transfer to Eppendorf tube. > Measure the A260 and calculate the concentration of DNA. > Ifnecessary (too diluted), add 40)11 of 3 M NH4Ac and 1 ml of ethanol. 1. 2 Transcription Reaction There are essentially two variations to this protocol: the transcripts can be made radioactive for direct visualization by gel electrophoresis, or they can be made cold, and then visualized by S1 analysis or other techniques.