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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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So, You have a mL or so of cells in a microfuge tube, harvest them in the cold and place on ice, and then aspirate the medium (aspiration works better than pipette and is faster). Go just above the pellet, but not TO the pellet. The liquid will rise up the wall the of the tube and get sucked away leaving a practically dry pellet. | So, You have a mL or so of cells in a microfuge tube, harvest them in the cold and place on ice, and then aspirate the medium (aspiration works better than pipette and is faster). Go just above the pellet, but not TO the pellet. The liquid will rise up the wall the of the tube and get sucked away leaving a practically dry pellet. | ||
Pipette a fixed defined volume of the lysis solution to each sample (generally 50-100 | 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 | 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 uL of linear polyacrylamide (LPA)at 10 mg/mL. | ||
===Organic Extraction and Precipitation=== | ===Organic Extraction and Precipitation=== | ||
