A Laboratory Guide to In Vitro Transcription by Dr. Felipe Sierra (auth.)

By Dr. Felipe Sierra (auth.)

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Stir in warm room (57 C) until dissolved, then add the other components. Store at _20DC at least overnight. This solution should be used very cold (not warmer than -10 C), because the lower temperatures help to increase its viscosity. 2 M sucrose and only 5% glycerol. 5 to 2 g/10 ml). PMSF (phenyl-methyl-sulfonyl-fluoride) is a Serine-protease inhibitor. It is soluble in isopropanol at room temperature. For storage, the solution is kept at -20 C, where the PMSF crystallizes. It can be redissolved by a brief heating at 57 C, and kept at room temperature until needed; if kept on ice, it will re-crystallize!

Spin at 9,000 rpm for 15 minutes, at room 56 III Experimental temperature or in the cold. Discard the supernatant. 5 ml of 10/2. > Add RNase A to 100 )1g/ml and RNase T 1 to 100 Units/ml. Incubate at 3rC for 30 minutes. 5m column. 1. > 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.

Spin at 9,000 rpm for 5 minutes in the cold. > Transfer the supernatant to a new tube containing 10 ml of isopropanol. Mix well and leave 5 minutes at room temperature. > Spin at 9,000 rpm for 15 minutes, at room 56 III Experimental temperature or in the cold. Discard the supernatant. 5 ml of 10/2. > Add RNase A to 100 )1g/ml and RNase T 1 to 100 Units/ml. Incubate at 3rC for 30 minutes. 5m column. 1. > Collect approximately 4 ml and discard. 7% agarose). 5 M. > Extract 3 times with phenol/chloroform.

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