Determine the OD600 and correlate the cell density from the chart. Set up four 100mL YPD cultures at the following densities: 0.7x105, 1x105, and 3.0x105 cells/mL
GROWTH OF CELLS
Grow 100mL of cells to OD600=0.7-0.8 at 23oC.
Add 5-10ul of BME, 15 minutes before spin.
Harvest cells in two 50mL conical tubes, spin 1.5-2.0K for 8-10min.
SPHEROPLASTING. Resuspend cells in 4mL YWB.
*NOTE: If cells have been arrested with nocodazole or alpha factor, include the inhibitor in the YWB. Let sit at room temperature for 2-3 min. Add oxalyticase [12uL 1mg/ml stock]. Incubate at 23oC with gentle shaking to form spheroplasts. Spheroplasting should be complete within one hour; avoid incubations longer than 1h, 15min
Transfer spheroplasted cells to a TOMY tube. Spin at 3.5K for 7min. Resuspend cells in 4mL YWB (containing 5% glycerol and 1mM PMSF [0.2M stock in 100% EtOH]) by gently pipetting up and down, using a 1mL pipet. Spin at 3.5K for 7min.
Gently resuspend cells in 2.5mL 1x EBB (containing 5% glycerol and 0.4mM PMSF).
Allow cells to "swell" for 10min. at room temperature, then transfer to homogenizer. [Rinse dounce with H2O, EtOH to clean. Rinse dounce with EBB before using.] Homogenize cells with 5 pestle strokes (up and down is one stroke). Avoid introducing air. Transfer to clean TOMY tube.
Add 150uL 5M NaCl (final concentration ~0.3M). Incubate at room temperature for 5 min.
Add 5mL 1x EBB-plus (containing 5% glycerol, 0.1mM DTT and 150 ug/mL BSA). Thus, final concentrations in 7.5mL lysate are 0.1M NaCl. Incubate at room temperature for 45 min.
Meanwhile, thaw microtubule "seeds" and put at 37oC for 30min. Add taxol to 10uM (e.g. 1ul 0.13mM taxol to 15ul MT seeds) and incubate an additional 15min.
Remove 250ul sample for TOTAL MATERIAL ("1T"). Keep this tube at room temperature, soas to be comparable to the other samples. Divide remaining lysate between eppendorfs tubes.
CLARIFY LYSATES
Spin eppendorf tubes at 15K for 20min. Pool supernatants in a 15mL conical tube and mix gently. Aliquot lysate to microfuge tubes (between 800ul and 1ml). Spin microfuge tubes at 15K for 20min.
Remove 250ul TOTAL SUPERNATANT ("1TS") to a fresh tube.
Remove 500ul clarified lysate from each microfuge tube to a fresh tube. Add 5ul 1mM taxol (in DMSO) to each tube - final concentration is 10uM taxol. Mix gently.
For a standard titration of binding activity, add microtubules to each tube in decreasing amount. Normally, 8, 4, 2, 1, 0.5 and 0uls (MTs made from ~3-6mg/ml PC bovine tubulin). For 0.5ul, make a 10-fold dilution of MTs in BRB80/30% glycerol buffer (containing 10uM taxol). Allow binding to proceed at room temperature for 15 min.
Pellet microtubule/minichromosome mix at 15K for 8 min. It should be possible to see the larger MT pellets.
Remove 250ul of each supernatant to fresh tube. These are the SUPERNATANT FRACTIONS. Aspirate all residual liquid with drawn out Pasteur pipets.
Resuspend MT pellets in 250ul of 1x EBB (containing 0.1mM DTT, 100mM NaCl, 5% glycerol and 1mg/ml BSA, but NO PMSF!). These are the PELLET FRACTIONS. NOTE: Ultimately, PELLETs will be 2x concentrated relative to SUPs.
Add 250ul of 2x ASSAULT buffer (containing tRNA and øX174) to all samples. Remove protein by adding 20uls of Proteinase K solution (15mg/ml from Boehringer Mannheim) to each tube. Incubate tubes at 50oC for ~1-1.5h (longer is better).
After Proteinase K treament, precipitate DNA by adding 250ul 6M NH4OAc and 700ml isopropanol. Store at -20oC overnight.
Spin down DNA at 15K, 30min. 4oC. Carefully aspirate or decant supernatants, removing as much supernatant as possible. NOTE: The "PELLET SAMPLE" precipitated pellets are often small. Wash all DNApellets with 100-200ul 70% ethanol. Spin briefly (~5min) and aspirate the ethanol supernatant. Allow DNApellets to air dry. Do not invert tubes.
Resuspend all DNA pellets in 30ul TE, incubating at room temperature or 4oC for several hours.
PREPARATION OF SAMPLES FOR SOUTHERN ANALYSIS.
Remove 15ul of each sample to a fresh tube. Add 1ul DNase-free RNase (Boehringer Mannheim, 1:1000 dilution of 2U/ul stock) to 1T, 1Ts, and all SUPERNATANT fraction. The PELLET fractions do not have to be "RNased". Incubate at room temperature for at least 30min. Add sample buffer to all tubes.
Load samples onto a 0.6% agarose gel containing EtBr at 0.2ug/ml. Include a small amount of supercoiled test plasmid (e.g. pDK370) in the DNA size standards to serve as a positive control for hybridization. The final concentration of supercoiled plasmid should be such that ~0.1ng is loaded. Run gel at 20 or 30V overnight.
Photograph gel. Note whether the intensities of the øX174 bands are even in all lanes and whether 2u circleDNA is visible in the SUPERNATANT fractions.
Process gel for transfer to GeneScreen Plus according to the Posiblot protocol. Transfer for at least 90 min. UV crosslink DNA to membrane. Air dry.
HYBRIDIZATION.
Prehybridize blot at 65oC for ~3h in Church buffer containing 0.5mg/ml denature salmon sperm DNA(usually 14ml Church buffer plus 0.7ml 10mg/ml SS DNA per blot). Add probe 105 cpms per lane) and hybridize at 65oC overnight (at least 18h). Wash blot 2x with Buffer 1, 15min each at 65oC and 2x with Buffer 2, 15min each at 65oC. Monitor the blot--the last wash may not be necessary. NOTE: Use the 0.9kb SmaI/PstI URA3 fragment from pDK377 to visualize pDK370 or other URA3-containing plasmids. Use the 1.8kb SalI/ClaI LEU2 fragment from pDK255 to visualize YCp41 or other LEU2-containing plasmids. Use the ~2kb XhoI/SalI fragment from CV13 to visualize endogenous 2ucircle DNA. Note: make up the YWB, EBB 2X assault buffer fresh each experiment.
