MINICHROMOSOME MICROTUBULE BINDING ASSAY



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.

YWB per 10ml
5mL 2M Sorbitol (if NZ arrested, add 40uL 1.5mg/mL
0.336mL 1M K2HPO4 N2 to 4mL YWB)
0.064mL 1M KH2PO4
4.6mL dH2O
  YWB, glycerol, PMSF
5mL 2M Sorbitol
0.336mL 1M K2HPO4
0.064mL 1M KH2PO4
0.5mL 100% ultrapure glycerol
0.05mL 0.2M PMSF
4.05mL dH2O
 
1X EBB, glycerol, PMSF (5mL)
1mL 5X EBB
0.25mL 100% glycerol
12.5uL 0.2M PMSF
3.75uL dH2O
  1X EBB, glycerol, DTT, BSA (10mL)
2mL 5X EBB
0.5mL glycerol
10uL 100mg/mL BSA
10uL 0.1M DTT
7.48mL dH2O
 
1X EBB, DTT, NaCl, BSA 5mL
1mL 5X Ebb
5uL 0.1M DTT
0.1mL 5M NaCl
50uL 100mg/mL BSA
0.25mL glycerol
3.6mL dH2O
2mL EBB
0.2mL NaCl
0.1 BSA
 
 


REAGENTS  
 
YWB:
50mL 2M sorbitol
16.8mL 0.2M K2HPO4
3.2mL 0.2M KH2PO4
30mL H2O
  0.1M DTT  
0.2M PMSF in ethanol or ispropanol   Glusulase  
5X EBB:
5mL 1M MgCl2
5mL TRIS-HCl, pH7.4
0.1mL 0.5M EDTA
90mL H2O
  5M NaCl  
1mM taxol in DMSO   2X ASSAULT BUFFER:
5mL 10% SDS, UltraPure
5mL 0.5M EDTA
5mL HEPES-KOH, pH 7.6 [7.5 w/NaOH]
85mL H2O
**2X A.B. can be frozen in 10mL aliquots.
[3.5mL Assault buffer, 0.5mL tRNA/øX174]
 
tRNA/øX174 DNA:
1mL 1mg/mL tRNA in H2O
0.1mL 10 ug/mL uncut øX174 DNA
  5X BRB80 100mLs 5X stock
80mM Pipes pH6.8(KOH) 10mL 800mM Pipes pH6.8 (KOH)
1mM EGTA 1ml 100mM EGTA
1mM MgCl2 0.1mL 1M MgCl2
 


MINICHROMOSOME-MICROTUBULE BINDING ASSAY


GROWTH OF CELLS. Grow 100mL of cells to OD600=0.7-0.8 at 23oC. For good binding activity it has proved important to maintain cells in log phase. Normally, 2 days prior to day of experiment, a single medium-sized cology is picked from selective medium and inoculated into 5mL YPS (or -URA, as I do). Two additional dilutions are made from this "neat" inoculum (e.g. 1:10 and 1:25). The goal is to have late log phase cultures the next day (~4x107 cells/mL).

The day prior to experiment, make a 1:10 dilution of the suitable 5mL overnight. Determine the OD600and correlating cell density from the chart. Set up three 100mL YPD cultures at the following densities: ~0.7x105, 1x105 and 1.5x105 cells/mL.




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