Hydroxyurea treatment of yeast cells with WT or deletion of MSH2 gene.

Materials

• 2M Hydroxy Urea (HU) stock solution in water: 5mL x 2M x FW 76.06/1000 = 0.7606 gram. 

HU are salt-like, and can dissolve in water in about 10 minutes at 30C with shaking. 

• 1 ug/ul Propidum Iodine(PrI) stock
• AGY124, yeast strain with pRS413 and pSH44 (This is the plasmid control)
• AGY125, yeast strain with the wild type pMSH2 and pSH44 (This is the wildtype MSH2 control)

These strains were shipped from Gammie lab in January 2015,
see http://hongqinlab.blogspot.com/2015/01/bio125-strains-from-gammie-lab-media.html

Grown in SD-HIS-TRP-URA media by Dr. Kioko on Monday March 16, 2015

• Microscope and smart-phone stages. Students can take pictures of cell morphology. 

Experimental procedure and results. 

Under microscope, WT and Vector control yeast cells are about 50% in budding phases (big cells with small buds). 

Vector control cells, about 50% in budding phases. 

WT in about 50% budding phases.

WT and Vec cells were treated with 200mM HU for 1 hour. Under microscope, it can be seen that HU-treated cells are most round and arrested at G1 phase. (Big balls in a string). 

For students:
Day1:
450ul log-culture +/- 50ul HU, monitor shape change under microscope.
After 1.5 hours, HU-arrested cells should be like watermelons.
Then wash with water once, resuspend with 500ul water, left at 4C.
(20160405. Water arrested cells just like HU, so cells ). 

Day 2.
spin down cells, add 500ul SD growth media,
shake at 30C
after 1.5 hours, monitor shape changes under microscope.
spin down, add 500ul 70% enthanol, shake at room temp

Day 2.5 Faculty do PI stain and flow cytomer run

Day 3. R exercise on flow data










References:
http://hongqinlab.blogspot.com/2013/12/sce-cell-cycle-and-morphology.html
http://hongqinlab.blogspot.com/2015/01/cell-cycle-and-msh2-project.html