Bonny Brewer's DNA isolation protocol for high-molecular weight yeast genomic DNA suitable for 2-D gel analysis of replication intermediates: A fusion of the "Huberman" and the "smash-n-grab" procedures.
|1.||Grow 300 ml of yeast culture to an OD of 0.5 to 0.7 in YComp. Add sodium azide to 0.1%. Quickly chill cells by addition to 40 ml of 0.2 M EDTA frozen at -20°C in 250 ml centrifuge bottles. (For 300 ml you will need two bottles.)|
|2.||Spin cells 5 min at 7000 rpm. Pour off medium, add 5 ml iced water to each bottle. Resuspend each cell pellet and decant into a 15 ml Falcon tube, pooling the two pellets back together. Spin down the cells in a swinging bucket centrifuge at 2500 rpm for 3 minutes. Pour off water and shake out loose water droplets. At this point the cell pellets can be frozen at -20°C.|
|3.||Add 0.2 ml NIB to the cell pellet and vortex briefly. Add 0.9 ml glass beads. There will be no obvious liquid above the level of the beads. Vortex at the highest rate with a very healthy vortexer, 5 to 10 times for 30 seconds, returning the cells to ice for 30 seconds between bursts of vortexing. Check for cell lysis by microscopy. Continue vortexing until desired percentage of broken cells is achieved.
NIB = Nuclear Isolation Buffer: 17% glycerol, 50 mM MOPS buffer, 150 mM potassium acetate, 2 mM magnesium chloride, 0.5 mM spermidine, and 0.15 mM spermine; pH is adjusted to 7.2 after all ingredients are dissolved.
|4.||Add 0.6 ml NIB to the broken cells, vortex briefly and remove the solution to an Eppendorf tube. Repeat with another 0.6 ml of NIB. There should now be ~1.3 ml transferred to the Eppendorf.|
|5.||Spin down nuclei, cell walls and unbroken cells in a microfuge for 20-30 minutes at top speed at 4°C. Pipette off the supernatant. Keep the pellet on ice.|
|6.||Resuspend the nuclei in 0.3 ml TEN (50 mM Tris, 50 mM EDTA, 100 mM NaCl) to which one microliter of RNAse A (10 mg/ml) has been added. Keep on ice 5 minutes.|
|7.||Add 12 microliters of 25% Sarkosyl. Invert tube gently a few times to mix. Add 1 microliter proteinase K (20 mg/ml). Invert tube again a few times to mix and transfer to 37°C for 30 minutes.|
|8.||Spin the lysed nuclei for 5 minutes at 4°C at top speed in a microfuge. Remove the supernatant with a pipette tip to a screw-cap Eppendorf tube. Add 0.3 ml phenol:chloroform:isoamyl alcohol (24:24:1). Invert a dozen times to mix the phases. Let it rest and repeat another dozen inversions. Spin the samples at top speed in a microfuge to separate the phases (the top aqueous phase should be clear).|
|9.||Cut off the tip of a 200 µl pipette tip to increase the diameter of the opening and carefully remove the aqueous phase to a clean eppendorf tube. It will take several attempts to recover all of the aqueous phase. Try not to take any of the white interphase.|
|10.||Estimate the aqueous phase that you have recovered (~0.25 ml is a rough estimate). Add 2 volumes of absolute ethanol that contains 0.5 M potassium acetate. Slowly and gently mix the phases by inverting the tube. The DNA should spool. When all of the aqueous phase has been incorporated into the alcohol phase, the DNA spool should collapse and fall to the bottom of the tube. Pull off the alcohol with a pipette tip and discard. To the DNA pellet, add 0.3 ml 70% ethanol. Spin 5 minutes at top speed. Aspirate the ethanol, invert the tube and let it air dry for 30 minutes.|
|11.||Resuspend the DNA in 75 µl (or so). It will probably take the DNA overnight to completely go into solution. Be aware that there may be RNA nucleotides in the solution so don't trust an OD260 reading. Run a microliter on a gel to confirm the DNA concentration and size.|