Tim Burland (Last Update September 21, 1993)
Thanks to many researchers who have contributed to this method, and a special thanks to Joyce Mohberg whose dependable method for isolating nuclei has endured with few changes for over three decades
Add 0.5-2ml exponentially growing microplasmodia to 40ml fresh SDM in a 500-ml flask. Shake vigorously (either use a baffled flask for gyrotary shakers or use a reciprocating shaker) @ 26o
48hrs later, wet weight should be 40-70mg/ml. Spin down by brief centrifugation in 50-ml conical centrifuge tubes (Corning #25335-50), wash once in 40ml sterile water (to remove slime), then resuspend in 40ml ice-cold NHS + 12ml Percoll (Sigma Cat # P1644). Mix. Keep on ice
Transfer to 500-ml ice-cold metal Waring blender cups, then blend at 65V (140V switch on rheostat) for 30sec. (For smaller volumes, use the 10-ml Sorvall Omnimixer cups at setting #4 for 60sec.)
Check a sample of the homogenate microscopically for complete homogenization without disruption of nuclei. If large lumps of material remain, homogenize further.
Let froth settle for about 5min. on ice
Transfer homogenates to 50-ml conical centrifuge tubes (Corning #25335-50) and centrifuge at 0o in the Sorvall H6000A rotor for 12min at 2000rpm. Slime should be forced to top of tube.
ASPIRATE off the slime and the supernatant. Put tube on ice.
Resuspend nuclei by vortexing in the small residue of liquid remaining in the tube, add ice-cold NHS to 45ml, mix, and centrifuge at 2000rpm for 10min at 0o. Pour off supernatant.
Wash nuclei once more in 45ml ice-cold NHS.
Resuspend nuclear pellet by vortexing in residual liquid on ice.
Mix lysis buffer with 1/20th vol proteinase K stock. Then add 4ml [lysis buffer+proteinase K] and IMMEDIATELY transfer to a 70o water-bath. Incubate 15min.
MIX GENTLY - DO NOT VORTEX.
Cool to 37o, add another 1/20th vol. proteinase K, & incubate overnight @ 37o
Add 1/10th vol RNAse solution and incubate 37o for 2hr
Phenol and chloroform are extremely dangerous chemicals. For use only by trained personnel in appropriate laboratory facilities
Extract once each with an equal volume of (i) buffer-saturated phenol, (ii) a 1:1 mix of buffer-saturated phenol : chloroform/isoamyl alcohol (24/1), and (iii) chloroform/isoamyl alcohol (24/1) (all room temp). If the phenol-chloroform extraction leaves a large mess of material at the aqueous-organic interface, do a second phenol-chloroform extraction before the chloroform extraction.
At room temp., add one fifteenth vol 3M sodium acetate then 2 volumes of ethanol & mix gently. Ppte should appear in a few seconds (if it does not, try incubating at -20o for 30 min).
Retrieve ppte - If ppte is stringy and in one piece, pluck it out with a sterile Dispo pipette, or pour off soup holding ppte in place with a pipette. Otherwise, centrifuge 2min @ full speed in a benchtop centrifuge.
Wash ppte x2 in 70% ethanol. Drain well in air to dry off the ethanol and most - but not all - of the moisture.
Resuspend DNA in 500ml TE. Dissolution is slow - heat to 65o for an hour if in a hurry.
Grow microplasmodia as described above
Spin down exponentially growing microplasmodia and resuspend in 0.5-1 Vol. SDM
Inoculate microplasmodia onto S&S 576 filters over SDM and grow overnight 26°
For maximum & representative DNA yield, ensure surface plasmodia are at MII + >2hrs by examining ethanol-fixed smears under phase microscope.
Then use a spatula to scrape plasmodia off filters, and transfer plasmodia quickly to ice-cold NHS in a blender cup. Use at least 35ml NHS for every plasmodium grown on a 9-cm dish.
Maintain culture in exponential growth.
Harvest 5-10x108 amoebae by centrifugation at full speed for 2min in a benchtop centrifuge.
Wash once in an equal volume of BSS
Resuspend amoebae in residual liquid, cool on ice, then add ice-cold NHS to resuspend cells at about 1-2x107/ml.
Harvest growing amoebae in water and subculture with live bacteria onto SM plates at 5x105 amoebae/plate. Incubate 26o for 2 days.
Harvest 5-10x108 amoebae in water, spin down 2min full speed in a benchtop centrifuge, and wash twice in water to remove bacteria.
Resuspend cells in water and make sure the ratio of amoebae:bacteria is less than 1:1 (if not, wash further in water). Spin down amoebae again.
Resuspend amoebae in residual liquid, then add NHS so that the cell concentration would be 1-2x107/ml.