Difference between revisions of "Growth Curve Experiment"

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Here's a brief protocol for monitoring yeast growth.
Here's a brief protocol for monitoring yeast growth.


*'''GROWTH CURVE'''
'''GROWTH CURVE'''
(1) Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain)  
(1) Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain)  
     (a) please be conservative with YPD media here - we can do these experiments in 100mL         
     (a) Streak Carolina strain on a YPD + Ura plate for isolated colonies.
          tubes - in triplicate. Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours.  
    (b) Grow overnight at 30C
    (c) Pick a single colony from the plate (in triplicate) and grow in 5mL YPD + Ura
    (d) The next day, Record the OD (650nm) of the >16hour culture, and dilute the Growth Curve Culture by adding 100uL of 16hour culture to 99.9mL of YPD + Ura.
    (e) Grow at 30C, 250rpm.
    (e) Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours. Use YPD + Ura as the blank for each reading.
 


(2) Repeat growth curve with pD1214:FAKS-hKappa cloned into yeast strain.
(2) Repeat growth curve with pD1214:FAKS-hKappa cloned into yeast strain.
     (a) no, it's not the real human kappa casein, but it'll get us closer to understanding how our strains will perform after real cloning happens.  
     (a) no, it's not the real human kappa casein, but it'll get us closer to understanding how our strains will perform after real cloning happens.  
           This is a great set of experiments for folks who've not done cloning before. Please learn about Bacterial transformation
           This is a great set of experiments for folks who've not done cloning before. Please learn about '''Bacterial transformation
           and plating for single colonies, Plasmid Prep, and Yeast transformation.
           and plating for single colonies, Plasmid Prep, and Yeast transformation'''.
     (b) grow the yeast clones in triplicate (100mL) to create growth curve.
     (b) Transform positive plasmid into E. coli (see Molecular Biology page)
 
    (c) Instead of plating transformation, add the transformation reaction to 5mL of LB + Carb(50).
 
    (d) Grow this plasmid overnight at 37C, rotating; and plasmid prep the DNA the next day.
Select a single colony and grow it overnight in 5mL YPD at 30C, rotating at 250rpm. Read the OD the next morning (>16hrs later). Record the OD at 16hours, then start the culture with a KNOWN amount of 16-hr innoculum.  
    (e) Transform yeast using the Yeast Transformation protocol.
 
    (f) Plate transformation and incubate overnight at 30C.
      (a) Dilute the 16hour overnight in YPD, or 0.1mL into 99.9mL (1:1000) YPD.
    (g) Select single colonies and repeat from (1) - (c) ('''above''').


      (b) Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours.
(3) RECORD YOUR DATA, MAKE A FIGURE, and POST IT HERE.

Revision as of 16:13, 20 August 2014

Chemical transformation of yeast requires the organism to be in logarithmic growth phase, so, before we clone our casein genes into yeast, we need to make sure that we know how to grow our yeast to log Phase.

Here's a brief protocol for monitoring yeast growth.

GROWTH CURVE (1) Figure out growth curve of yeast strains (we have Carolina strain now, we want ATCC strain, and Keoni Strain) 

    (a) Streak Carolina strain on a YPD + Ura plate for isolated colonies.
    (b) Grow overnight at 30C
    (c) Pick a single colony from the plate (in triplicate) and grow in 5mL YPD + Ura
    (d) The next day, Record the OD (650nm) of the >16hour culture, and dilute the Growth Curve Culture by adding 100uL of 16hour culture to 99.9mL of YPD + Ura.
    (e) Grow at 30C, 250rpm.
    (e) Record the OD (650nm) at 0, 5, 10, 24, 48, and 72 hours. Use YPD + Ura as the blank for each reading.


(2) Repeat growth curve with pD1214:FAKS-hKappa cloned into yeast strain. 

    (a) no, it's not the real human kappa casein, but it'll get us closer to understanding how our strains will perform after real cloning happens. 
         This is a great set of experiments for folks who've not done cloning before. Please learn about Bacterial transformation
         and plating for single colonies, Plasmid Prep, and Yeast transformation.
    (b) Transform positive plasmid into E. coli (see Molecular Biology page)
    (c) Instead of plating transformation, add the transformation reaction to 5mL of LB + Carb(50).
    (d) Grow this plasmid overnight at 37C, rotating; and plasmid prep the DNA the next day.
    (e) Transform yeast using the Yeast Transformation protocol.
    (f) Plate transformation and incubate overnight at 30C.
    (g) Select single colonies and repeat from (1) - (c) (above).

(3) RECORD YOUR DATA, MAKE A FIGURE, and POST IT HERE.

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