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Sauer:RNA Purification from E. coli: Difference between revisions
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Sauer:RNA Purification from E. coli (view source)
Revision as of 14:15, 24 January 2007
, 24 January 2007→For stable RNAs
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Pipette a fixed defined volume of the lysis solution to each sample (generally 50-100 μL) and vortex into solution at room temp. Place the tube in a rack at room temp and proceed to the next pellet. After resuspension, the cells will clear in less than a minute when placed at room temp. | Pipette a fixed defined volume of the lysis solution to each sample (generally 50-100 μL) and vortex into solution at room temp. Place the tube in a rack at room temp and proceed to the next pellet. After resuspension, the cells will clear in less than a minute when placed at room temp. | ||
So now you have intact ribosomes in a low-salt solution. Add about 200-400 uL of salty extraction buffer. This solution chelates all of the Mg++, disrupts ribosomes, increases ionic strength, and provides a carrier for precipitation. I use 50 mM bis-Tris pH 6.5, containing 400 mM NaCl, 5 mM EDTA and 1 μL/400 | So now you have intact ribosomes in a low-salt solution. Add about 200-400 uL of salty extraction buffer. This solution chelates all of the Mg++, disrupts ribosomes, increases ionic strength, and provides a carrier for precipitation. I use 50 mM bis-Tris pH 6.5, containing 400 mM NaCl, 5 mM EDTA and 1 μL/400 μL of linear polyacrylamide (LPA)at 10 mg/mL. | ||
===Organic Extraction and Precipitation=== | ===Organic Extraction and Precipitation=== | ||
